Method for identifying tissue plasminogen activator having zymogenic or fibrin specific properties

ABSTRACT

Tissue plasminogen activator (t-PA) zymogens and variants are prepared, including a fibrinolytically active variant of t-PA that has an amino acid alteration at a site within the protease domain of t-PA as compared with the corresponding wild-type t-PA, which alteration renders the variant zymogenic in the presence of plasmin-degraded fibrinogen, and/or fibrin (or plasma clot) specific, as compared to the corresponding wild-type t-PA. DNA sequences can be prepared that encode the zymogens and variants, as well as expression vectors incorporating the DNA sequences, and host cells transformed with the expression vectors. The zymogens and variants may be used in a pharmaceutical preparation to treat a vascular disease or condition or to prevent fibrin deposition or adhesion formation or reformation in mammals.

This is a continuation of application Ser. No. 08/088,451 filed on 06Jul. 1993, which is a continuation of application Ser. No. 07/770,510filed 03 Oct. 1991, now U.S. Pat. No. 5,262,170, which is a continuationof application Ser. No. 07/384,608 filed 24 Jul. 1989, now U.S. Pat.5,108,901, which in turn, is a continuation-in-part of Ser. No.07/240,856 filed 02 Sep. 1988, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to particular tissue plasminogenactivator (t-PA) zymogens, to methods for preparing such zymogens, andto methods and compositions utilizing such zymogens in pharmaceuticalapplications. In addition, the invention relates to variants having amodified structure that includes substituted amino acids within theprotease domain of t-PA, which modification renders the variantzymogenic, i.e., relatively inactive in its one-chain form but activewhen converted to its two-chain form in the presence of fibrin, and/ormore fibrin (or plasma clot) specific than wild-type (wt) t-PA.

2. Description of Background and Related Art

Plasminogen activators are enzymes that activate the zymogen plasminogento generate the serine proteinase plasmin (by cleavage at Arg561-Val562)that degrades various proteins, including fibrin. Among the plasminogenactivators studied are streptokinase, a bacterial protein, urokinase, anenzyme synthesized in the kidney and elsewhere and originally extractedfrom urine, and human tissue plasminogen activator (t-PA), an enzymeproduced by the cells lining blood vessel walls.

The mechanism of action of each of these plasminogen activators differs:Streptokinase forms a complex with plasminogen or plasmin, generatingplasminogen-activating activity, urokinase cleaves plasminogen directly,and t-PA, fibrin, and plasminogen all interact to yield maximumactivity.

t-PA has been identified and described as a particularly important andpotent new biological pharmaceutical agent that has shown extraordinaryresults in the treatment of vascular diseases, such as myocardialinfarction, due in part to its high fibrin specificity and potentability to dissolve blood clots in vivo.

Although the existence of t-PA prompted numerous investigations byseveral scientific groups, it was first identified as a substantiallypure isolate from a natural source, and tested for requisite plasminogenactivator activity in vivo, by Collen et al., U.S. Pat. No. 4,752,603issued Jun. 21, 1988. See also Rijken et al., J. Biol. Chem., 256: 7035(1981).

Subsequently, t-PA was fully identified and characterized by underlyingDNA sequence and deduced amino acid sequence based on successful workemploying recombinant DNA technology resulting in large quantities oft-PA in a distinct milieu. This work was reported by Pennica et al.,Nature, 301: 214 (1983)) and in U.S. Pat. No. 4,766,075, issued 23 Aug.1988.

Based on these disclosures, it seems now clear that the t-PA moleculecontains five domains that have been defined with reference tohomologous or otherwise similar structures identified in various otherproteins such as trypsin, chymotrypsin, plasminogen, prothrombin,fibronectin, and epidermal growth factor (EGF). These domains have beendesignated, starting at the N-terminus of the amino acid sequence oft-PA, as 1) the finger region (F) that has variously been defined asincluding amino acids 1 to about 44, 2) the growth factor region (G)that has been variously defined as stretching from about amino acids 45to 91 (based upon its homology with EGF), 3) kringle one (K1) that hasbeen defined as stretching from about amino acid 92 to about amino acid173, 4) kringle two (K2) that has been defined as stretching from aboutamino acid 180 to about amino acid 261, and 5) the so-called serineprotease domain (P) that generally has been defined as stretching fromabout amino acid 264 to the C-terminal end of the molecule. Thesedomains, which are situated generally adjacent to one another, or areseparated by short "linker" regions, account for the entire amino acidsequence of from 1 to 527 amino acids of the putative mature form oft-PA.

Each domain has been described variously as contributing certainspecific biologically significant properties. The finger domain has beencharacterized as containing a sequence of at least major importance forhigh binding affinity to fibrin. (This activity is thought important forthe high specificity that t-PA displays with respect to clot lysis atthe locus of a fibrin-rich thrombus.) The growth factor-like regionlikewise has been associated with cell surface binding activity. Thekringle 2 region also has been strongly associated with fibrin bindingand with the ability of fibrin to stimulate the activity of t-PA. Theserine protease domain is responsible for the enzymatic cleavage ofplasminogen to produce plasmin.

Despite the profound advantages associated with natural t-PA as aclot-dissolving agent, it is not believed that the natural proteinnecessarily represents the optimal t-PA agent under all circumstances.Therefore, several variants have been proposed or devised to enhancespecific properties of t-PA. Certain of those variants addressdisadvantages associated with the use of natural t-PA in situationswhere an agent with a longer half-life or slower clearance rate would bepreferred, e.g., in the treatment of deep-vein thrombosis and followingreperfusion of an infarct victim, or where a single-chain agent ispreferred.

For example, removal of a substantial portion or all of the fingerdomain results in a molecule with substantially diminished fibrinbinding characteristics, albeit in return there is a decrease in theoverall rate of clearance of the resultant entity--See WO 89/00197published 12 Jan. 1989.

Variants are described in EPO Pat. Publ. No. 199,574 that have aminoacid substitutions at the proteolytic cleavage sites at positions 275,276, and 277. These variants, characterized preferentially as t-PAvariants having an amino acid other than arginine at position 275, arereferred to as protease-resistant one-chain t-PA variants in that,unlike natural t-PA, which can exist in either a one-chain or two-chainform, they are resistant to protease cleavage at position 275 and aretherefore not converted metabolically in vivo into a two-chain form.This form is thought to have certain advantages biologically andcommercially, in that it is more stable while the fibrin binding andfibrin stimulation are increased relative to two-chain t-PA.Furthermore, plasminogen activators are described that comprise onedomain capable of interacting with fibrin and the protease domain ofurokinase, with one embodiment being urokinase altered to make it lesssusceptible to forming two-chain urokinase. See WO 88/05081 published 14Jul. 1988.

For further patent literature regarding modification of the proteasecleavage site of t-PA, see, for example, EPO Pat. Nos. 241,209; EP201,153 published Nov. 12, 1986; EP 233,013 published Aug. 19, 1987; EP292,009 published Nov. 23, 1988; EP 293,936 published Dec. 7, 1988; andEP 293,934 published Dec. 7, 1988; and WO 88/10119.

Glycosylation mutants at 117-119, 184-186, and 448-450 exhibited higherspecific activity as the mole percent carbohydrate was reduced. See EPOPub. No. 227,462 published Jul. 1, 1987. This patent applicationadditionally discloses using an assay of fibrin/fibrin degradationproducts and teaches that one may also modify the t-PA molecule atpositions 272-280 or delete up to 25 amino acids from the C-terminus.Further, the t-PA mutants with Asn119, Ala186 and Asn450, which have theN-glycosylation sites selectively removed by DNA modification butcontain residual O-linked carbohydrate, were found to be about two-foldas potent as melanoma t-PA in an in vitro lysis assay. See EPO Publ. No.225,286 published Jun. 10, 1987.

Replacement of the amino acid at 449 of t-PA with any amino acid exceptarginine to modify the glycosylation site, as well as modification ofArg275 or deletion of the -3 to 91 region, is also taught. See WO87/04722 published Aug. 13, 1987. An amino acid substitution at position448 of t-PA is disclosed as desirable to remove glycosylation. See EPOPub. No. 297,066 published Dec. 28, 1988. The combination ofmodifications at positions 448-450 and deletion of the N-terminal 1-82amino acids is disclosed by WO 89/00191 published Jan. 12, 1989.Additionally, urokinase has been modified in the region ofAsp302-Ser303-Thr304 to prevent glycosylation. See EPO Pub. No. 299,706published 18 Jan. 1989.

However, alteration of the glycosylation sites, and in particular thatat amino acid 117, seems invariably to result in a molecule havingaffected solubility characteristics that may result additionally in analtered circulating half-life pattern and/or fibrin bindingcharacteristics. See EPO Pat. Publ. No. 238,304, published 23 Sep. 1987.

When the growth factor domain of t-PA is deleted, the resultant variantis still active and binds to fibrin, as reported by A. J. van Zonneveldet al., Thrombos. Haemostas., 54 (1) 4 (1985). Various deletions in thegrowth factor domain have also been reported in the patent literature.See EPO Publ. No. 241,209 (des-51-87), EPO Publ. No. 241,208 (des-51-87and des-51-173), PCT 87/04722 (deletion of all or part of the N-terminal1-91), EPO Publ. No. 231,624 (all of growth factor domain deleted), andEPO Publ. No. 242,836 and Jap. Pat. Appl. Kokai No. 62-269688 (some orall of the growth factor domain deleted).

It has further been shown that t-PA can be modified both in the regionof the first kringle domain and in the growth factor domain, resultingin increased circulatory half-life. See EPO Pat. Publ. No. 241,208published Oct. 14, 1987. The region between amino acids 51 and 87,inclusive, can be deleted from t-PA to result in a variant having slowerclearance from plasma. Browne et al., J. Biol. Chem., 263: 1599-1602(1988). Also, t-PA can be modified, without adverse biological effects,in the region of amino acids 67 to 69 of the mature, native t-PA, bydeletion of certain amino acid residues or replacement of one or moreamino acids with different amino acids. See EPO Pat. Publ. No. 240,334published Oct. 7, 1987.

A hybrid of t-PA/urokinase using the region of t-PA encompassing aminoacids 273-527 is also disclosed. See EPO 290,118 published Nov. 9, 1988.

Serpin-resistant mutants of human t-PA with alterations in the proteasedomain, including d296-302 t-PA, R304S t-PA, and R304E t-PA, aredisclosed in Madison et al., Nature, 339: 721-724 (1989); see also theaccompanying article by Dagmar Ringe in the same issue.

A general review of plasminogen activators and second-generationderivatives thereof can be found in Harris, Protein Engineering, 1:449-458 (1987). Other reviews of t-PA variants include Pannekoek et al.,Fibrinolysis, 2: 123-132 (1988) and Ross et al., in Annal Reports inMedicinal Chemistry, Vol. 23, Chapter 12 (1988).

While the foregoing disclosures provide evidence that newer and, invarious respects, better t-PA agents are at hand, there are currently not-PA molecules described that only become activated when they reach thesite of the clot to be dissolved. Currently, the t-PA molecules areactive in the presence of fibrin and/or plasma proteins or whole blood,whether they are in the one-chain or two-chain form. It would bedesirable to have a zymogenic t-PA that in the presence of fibrinrequires clipping of its one-chain form to its two-chain form to becomefully active. Such variant molecules would likely exhibit fewer sideeffects, such as less bleeding, and have fibrinogen sparing properties,thereby providing medical science important new alternatives in thetreatment of cardiovascular disease and numerous other medicalconditions that arise from thromboembolic occlusion of blood vessels, aswell as in the prevention of the formation of adhesions.

It would also be desirable to provide a t-PA molecule that, relative towild-type t-PA, has a higher fibrin-stimulated (or a plasmaclot-stimulated) activity than fibrinogen-stimulated (orplasma-stimulated) activity, i.e., is fibrin (or plasma clot) specific,so that it will act only at the site of the clot and not systemically.

Accordingly, it is an object of this invention to provide zymogenicand/or fibrin-specific t-PA molecules that exhibit improved therapeuticand pharmaceutical characteristics.

It is another object to provide for the treatment of conditions thatadmit the use of clot-dissolving agents that are active only at the siteof the clot and are useful at higher levels than other such agents.

These and other objects will be apparent to one of ordinary skill in theart.

SUMMARY OF THE INVENTION

These objects are achieved by the provision of a tissue plasminogenactivator (t-PA) zymogen capable of converting to the enzymaticallyactive form of t-PA upon cleavage by plasmin. In another aspect, theinvention provides a t-PA variant having an amino acid alteration at asite or sites within the protease domain of t-PA as compared with thecorresponding wild-type t-PA, which alteration renders the variantzymogenic as compared to the corresponding wild-type t-PA.

In one particularly preferred embodiment, the t-PA is human t-PA and thealteration is in the region of 305, inclusive, such as a substitution ofhistidine for phenylalanine at position 305 of the correspondingwild-type t-PA.

In other embodiments, this invention relates to a DNA sequence encodingthe zymogen and variant described above, replicable expression vectorscapable of expressing the DNA sequence in a transformant host cell, andmicroorganisms and cell cultures transformed with the vector.

In still another embodiment, the invention provides a method comprising:

(a) introducing an amino acid variation into the protease domain oft-PA; and

(b) screening the resultant t-PA variant for zymogenic character.

In other aspects, the invention supplies a human tissue plasminogenactivator (t-PA) variant capable of exhibiting one or more of thefollowing biological activities: zymogenic activity, fibrin specificity,or plasma clot specificity, characterized in that it contains an aminoacid alteration in its protease domain as compared with thecorresponding wild-type t-PA, which alteration is responsible for saidbiological activity, provided that such alteration excludes alterationssolely in the regions of 270-280, 448-450, and 502-527. Preferably, thevariant is such that the alteration is a substitution.

In another aspect, the invention provides a method comprising:

(a) introducing an amino acid variation into the protease domain oftissue plasminogen activator (t-PA); and

(b) screening the resultant t-PA variant for its capability ofexhibiting one or more of the following biological activities: zymogenicactivity, fibrin specificity, or plasma clot specificity.

In further embodiments the invention provides DNA sequences andreplicable vectors encoding the above-described variants and host cellstransformed with them.

In yet another embodiment, the invention is directed to a compositionfor treating a vascular condition or disease comprising atherapeutically effective amount of the zymogen or variant herein inadmixture with a pharmaceutically acceptable carrier. Also encompassedherein is a composition for preventing fibrin deposition or adhesionformation or reformation comprising a therapeutically effective amountof the zymogen or variant herein in admixture with a pharmaceuticallyacceptable carrier.

In still another embodiment, the invention provides a method of treatinga vascular condition or disease in a mammal comprising administering aneffective amount of the appropriate composition described above to themammal.

The invention also provides a method of treating a mammal to preventfibrin deposition or adhesion formation or reformation comprisingadministering to a site on the mammal of potential fibrin or adhesionformation an effective amount of the appropriate composition describedabove.

The first aspect of the present invention is based, inter alia, uponspecific successful research demonstrating that certain t-PA moleculesare zymogens in the presence of a stimulator of t-PA activity, such asplasmin-degraded fibrinogen fragments, and thus can have theirfibrinolytic activity turned off when generally in the plasma andactivated when proximate to plasmin at the site of the clot. Thus, thezymogen is activated on demand for specific localized clot therapy. Thezymogens herein are expected to be fibrinogen sparing and are generallyuseful in higher doses than their non-zymogenic counterparts, resultingin faster clot lysis and lysis of more clots.

The second aspect of this invention is to obtain a t-PA molecule that ismore fibrin (or plasma clot) specific so that it will act morepreferentially at the site of the clot than unmodified t-PA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the primary structure of t-PA showing the location of thefive domains, the disulfide bridging, and the activation site where themolecule is clipped into a two-chain molecule.

FIGS. 2 and 3 are schematic representations of a suitable method for thepreparation of pCISt-PA, together with a description of certain of itsprominent restriction sites.

FIG. 4 is a schematic representation of a suitable method for thepreparation of p7-1H, together with a description of certain of itsprominent restriction sites.

FIG. 5 shows fibrin binding of predominantly one-chain F305H (closedtriangles), two-chain F305H (closed circles with double lines),one-chain wild-type t-PA (closed squares), two-chain wild-type t-PA(closed diamonds), and a mixture of one-chain and two-chain wild-typet-PA (open triangles), at a t-PA concentration of 10 ng/ml.

FIGS. 6-9 show plots of the kinetics of the conversion of plasminogen toplasmin in the presence of plasmin-degraded fibrinogen by thepredominantly one-chain wild-type t-PA (FIG. 6), the two-chain wild-typet-PA (FIG. 7), the predominantly one-chain F305H t-PA (FIG. 8), and thetwo-chain F305H t-PA (FIG. 9). The squares, lines, and circles representvarious concentrations of plasminogen and t-PA in the assay buffer whichare specified at the end of Example I. In these Figures, the ordinate isthe absorbance at 405 nm and the abscissa is the square of the number ofminutes at which the absorbance was taken.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions

As used herein, the terms "human tissue plasminogen activator," "humant-PA," and "t-PA" denote human extrinsic (tissue-type) plasminogenactivator having two functional regions consisting of a protease domainthat is capable of converting plasminogen to plasmin and an N-terminalregion believed to be responsible for fibrin binding. These three termstherefore include polypeptides containing these functional domains aspart of the overall sequence. t-PA is suitably produced, e.g., byrecombinant cell culture systems, in bioactive forms comprising theprotease portion and portions of t-PA otherwise native to the source ofthe t-PA. It will be understood that natural allelic variations existand occur from individual to individual, demonstrated by (an) amino aciddifference(s) in the overall sequence.

As used herein, the term "wild-type t-PA" refers to the t-PA encoded bythe cDNA reported by U.S. Pat. No. 4,766,075, supra, the disclosure ofwhich is incorporated herein by reference. The t-PA thus encoded issuitably a t-PA molecule from any native source or any recombinantexpression system, including 293 or 294 cells, Chinese hamster ovarycells, etc.

As used herein, the term "protease domain" refers to the region of themature form of wild-type t-PA from amino acid 264 to amino acid 527,inclusive.

As used herein, the terms "zymogen," "zymogenic," and "zymogenicactivity" used to describe the t-PA herein must meet either one or bothof the definitions given below. In the first definition, these termssignify that in the presence of plasmin-degraded fibrinogen the t-PArequires clipping of its one-chain form to its two-chain ("enzymaticallyactive") form, as occurs in the presence of plasmin, to increase itsenzymatic activity, as defined below, under the conditions of the assaydescribed below.

In the presence of fibrinogen fragments, the one-chain form of the t-PA(zymogen) as defined herein is less active, as measured by the assaydescribed below, than wild-type two-chain t-PA and is converted to itsmore enzymatically active form when activated by exposure to a level ofplasmin that effects complete conversion of the single-chain form to thedouble-chain form. In general, the activity of the one-chain form isreduced to 50% or less of the activity of the corresponding two-chainform, preferably to 20% or less, and more preferably to less than 10% ofthe activity; and, upon clipping to the two-chain form, the activity isincreased to from about 20 to over 100%, preferably to at least 50%, ofthe activity of the wild-type two-chain form.

The variant is assayed for its enzymatic activity by determining thekinetics of conversion of plasminogen to plasmin using the chromogenicplasmin substrate S-2251 in the presence of fibrinogen fragments, usingthe assay described in Example I below.

For purposes herein regarding the first definition of a zymogen, azymogen is one that under the above conditions exhibits a distinct lagin the plasmin production, and thus in the increase in A405 when plottedvs. time², yet exhibits linear kinetics with increasing time. Adescription of time² kinetics can be found in Nieuwenhuizen, W.,Voskuilen, M., Traas, D., Hoegee-de Nobel, B., Verheijen, J. H., inFibrinogen-Structural Variants and Interactions, eds. A. Henschen, B.Hessel, J. McDonagh, T. Saldeen (1985), p. 331-342, the disclosure ofwhich is incorporated by reference. Without being limited to any onetheory, this effect is presumably due to the plasmin-catalyzedconversion of the one-chain to two-chain form, thereby leading toactivation of the t-PA zymogen. This contrasts with the observance oflinear kinetics from the beginning of the assay for the wild-typeone-chain t-PA, the wild-type two-chain t-PA, and the two-chain form ofthe zymogenic t-PA.

In the second, alternative definition, a "zymogen" specifically refersto a t-PA molecule that in an assay of enzymatic activity exhibits alarger differential activity between the one-chain form and thetwo-chain form than wild-type recombinant t-PA (rt-PA). The differentialactivity of the zymogen is preferably at least approximately 1.5 timesthat of wild-type rt-PA. This activity can be obtained by lowering theactivity of the one-chain form to a greater extent than that of thetwo-chain form relative to wild-type rt-PA; raising the activity of thetwo-chain form to a greater extent than that of the one-chain formrelative to wild-type rt-PA; or any combination of events describedabove that yield the described effect. The zymogenic character ofwild-type t-PA is described in Loscalzo, J. Clin. Invest., 82:1391-1397(1988) and Ranby et al., Thrombosis Research, 27: 175-183 (1982).

The expression "fibrin specificity" refers to the activity of a mutantthat exhibits a higher ratio of fibrin-dependent specific activity tofibrinogen-dependent specific activity in a S-2251 assay (in either theone-chain or two-chain form) than wild-type rt-PA, and preferably aratio of at least 1.5.

The expression "plasma clot specificity" refers to the activity of amutant that exhibits a higher ratio of plasma clot-dependent specificactivity to plasma-dependent specific activity in a S-2251 assay (ineither the one-chain or two-chain form) than wild-type rt-PA, andpreferably a ratio of at least 1.5.

As used herein, "transient expression system" denotes a cell culturecontaining cells transfected with a t-PA variant-encoding vector thatexpresses the DNA sequence encoding the variant transiently, i.e., in amanner that may not be stable. Such cells are deemed "capable oftransient expression."

B. General Methods

1. Amino Acid Sequence Variants

For purposes of discussing the variants herein, reference is made toFIG. 1, which illustrates the primary structure of t-PA.

In FIG. 1, the letters in the circles are single-letter amino acidcodes, the connecting lines between chains indicate disulfide bridging,the open circles indicate glycosylation sites, and the designations F,GF, K1, K2, and SP indicate, respectively, the finger, growth factor,kringle 1, kringle 2, and serine protease domains.

For purposes of shorthand designation of t-PA variants described herein,it is noted that numbers refer to the amino acid residue/position alongthe amino acid sequences of putative mature t-PA (EPO Publ. No. 93,619).Amino acid identification uses the single-letter alphabet of aminoacids, i.e.,

    ______________________________________                                        Asp     D      Aspartic acid                                                                             Ile   I    Isoleucine                              Thr     T      Threonine   Leu   L    Leucine                                 Ser     S      Serine      Tyr   Y    Tyrosine                                Glu     E      Glutamic acid                                                                             Phe   F    Phenylalanine                           Pro     P      Proline     His   H    Histidine                               Gly     G      Glycine     Lys   K    Lysine                                  Ala     A      Alanine     Arg   R    Arginine                                Cys     C      Cysteine    Trp   W    Tryptophan                              Val     V      Valine      Gln   Q    Glutamine                               Met     M      Methionine  Asn   N    Asparagine                              ______________________________________                                    

The designation for a substitution variant herein consists of a letterfollowed by a number followed by a letter. The first (leftmost) letterdesignates the amino acid in the wild-type, mature t-PA. The numberrefers to the amino acid position where the amino acid substitution isbeing made, and the second (right-hand) letter designates the amino acidthat is used to replace the wild-type amino acid. The designation for aninsertion variant consists of the letter i followed by a numberdesignating the position of the residue in wild-type, mature t-PA beforewhich the insertion starts, followed by one or more capital lettersindicating, inclusively, the insertion to be made. The designation for adeletion variant consists of the letter d followed by the number of thestart position of the deletion to the number of the end position of thedeletion, with the positions being based on the wild-type, mature t-PA.Multiple mutations are separated by a comma in the notation for ease ofreading them.

Examples of the nomenclature are as follows: a substitution variantwhere the phenylalanine at position 305 of the wild-type t-PA isreplaced with a histidine residue is designated F305H. A substitutionvariant with multiple substitutions at consecutive positions 296-299 ofAAAA for KHRR is designated K296A, H297A, R298A, R299A. An insertionvariant where cysteine and valine are inserted after position 305 ofwild-type t-PA is designated i305CV. A deletion variant where the aminoacids at positions 300 to 305 are deleted from the wild-type, maturet-PA is designated d300-305. The notation `t-PA` follows after eachmutant.

One preferred class of zymogens herein are those that have asubstitution, deletion, or insertion in or around position 305 ofwild-type t-PA. These variants include those with an amino acid otherthan phenylalanine at position 305 of the corresponding wild-type t-PA.More preferably, such variants are those with an amino acid at position305 having a side chain that can act or does act as a hydrogen bonddonor, such as one containing a hydroxyl group or nitrogen atom. Stillmore preferably, such amino acids are arginine, lysine, tyrosine,asparagine, glutamine, and histidine, most preferably histidine.Preferred insertional zymogenic variants of this type include those withone or more, preferably one, amino acid inserted after the amino acid atposition 304 or 305, such as the amino acids described above, i.e.,those with a side chain that can acts or act as a hydrogen bond donor,such as one containing a hydroxyl group or nitrogen atom, e.g.,arginine, lysine, tyrosine, asparagine, glutamine, and histidine, mostpreferably histidine. Preferred deletion zymogenic variants includethose with deletions in the region of 297 to 305, inclusive, of thewild-type t-PA, including d297 t-PA, d298 t-PA, etc. and combinationsthereof, such as d297-299 t-PA or d297, d305 t-PA.

Particular embodiments of the above-noted zymogen variants include:F305H t-PA; F305T t-PA; F305N t-PA; F305K t-PA; F305R t-PA; F305Q t-PA;i304H t-PA; i304T t-PA; i304N t-PA; i304K t-PA; i304R t-PA; i304Q t-PA;i304HH t-PA; i305H t-PA; i305T t-PA; i305N t-PA; i305K t-PA; i305R t-PA;i305Q t-PA; i304H, i305H t-PA; i305HH t-PA; d297 t-PA; d298 t-PA; d299t-PA; d300 t-PA; d301 t-PA; d302 t-PA; d303 t-PA; d304 t-PA; d305 t-PA;d297-298 t-PA; d297-299 t-PA; d297-300 t-PA; d297-301 t-PA; d297-302t-PA; d297-303 t-PA; d297-304 t-PA; d297-305 t-PA; d300-301 t-PA;d300-302 t-PA; d300-303 t-PA; d300-304 t-PA; d300-305 t-PA; d304- 305t-PA; d297, d300 t-PA; d297, d305 t-PA; d1-44, N184D, F305H t-PA; d1-44,F305H t-PA; d1-44, I210R, G211A, K212R, V213R, F305H t-PA; d1-44, I210R,G211A, K212R, V213K, F305H t-PA; d1-44, V213K, F305H t-PA; d1-44, T252R,F305H t-PA; d1-44, V213K, T252R, F305H t-PA; d1-44, I210K, F305H t-PA;d1-44, I210R, G211H, K212Q, V213K, F305H t-PA; I210R, G211H, K212Q,V213K, F305H t-PA; I210R, G211A, K212R, V213R, F305H t-PA; d1-44, N184D,I210R, G211A, K212R, V213R, T252R, F305H t-PA; N184D, I210R, G211A,K212R, V213R, T252R, F305H t-PA; d92-179, F305H t-PA; d92-179, I210R,G211A, K212R, V213R, F305H t-PA; d92-179 , N184D, I210R, G211A, K212R,V213R, T252R, F305H t-PA; d92-179, I210R, G211A, K212R, V213R, T252R,F305H t-PA; Y67N, F305H t-PA; d1-44, Y67N, F305H t-PA; and the T252R orN184S analogues thereof or combinations thereof. (The changes other thanthose in the protease domain are described further below.)

Of these, the preferred zymogen variants are F305H t-PA; F305T t-PA;F305N t-PA; F305K t-PA; F305R t-PA; F305Q t-PA; i304H t-PA; d1-44, F305Ht-PA; d92-179, F305H t-PA; d1-44, N184D, F305H t-PA; d1-44, I210R,G211A, K212R, V213R, F305H t-PA; d1-44, I210R, G211A, K212R, V213K,F305H t-PA; I210R, G211A, K212R, V213R, F305H t-PA; d92- 179, I210R,G211A, K212R, V213R, F305H t-PA; d92-179, N184D, I210R, G211A, K212R,V213R, F305H t-PA; d92-179, N184D, I210R, G211A, K212R, V213R, T252R,F305H t-PA; d1-44, N184D, I210R, G211A, K212R, V213R, T252R, F305H t-PA;and N184D, I210R, G211A, K212R, V213R, T252R, F305H t-PA.

More preferred variants of the first class of zymogens are F305H t-PA;F305T t-PA; F305N t-PA; F305Q t-PA; i304H t-PA; d1-44, F305H t-PA;d92-179, F305H t-PA, and the most preferred F305H t-PA.

Another preferred class of zymogens herein, as well as a class ofvariants that may alternatively or additionally be fibrin (or plasmaclot) specific, are variants with one or more alterations in smallregions of the protease domain identified as having charged amino acidside chains, which regions and/or regions adjacent thereto may beresponsible for the interaction of t-PA with other substances that mightaffect its various activities.

The regions identified for testing for activity by this method are atresidue numbers 267, 283-287, 296-299, 303-304, 322, 326-327, 331-332,339-342, 347-351, 353-356, 360-362, 364-366, 369-371, 378-383, 387-392,400-405, 408, 410, 416-418, 426-430, 432-434, 440, 445-449, 449-453,460-462, 471-472, 477, 487-489, 505-506, 513, 519-523, and 523-526 ofthe corresponding wild-type t-PA.

One or more of these regions, or subunits thereof, are altered todetermine if the desired biological property or properties will beobtained. The charged residues (Arg, Asp, His, nys, and Glu) aresuitably identified using a technique known as alanine-scanningmutagenesis, disclosed in Cunningham and Wells, Science, 244:1081-1085(1989), the disclosure of which is incorporated herein by reference, andreplaced with a neutral or negatively charged amino acid to affect theinteraction of the amino acids with the surrounding aqueous environmentin or outside the cell.

The mutants found to be in this second preferred class of zymogens andfibrin-specific molecules are those wherein the amino acids replaced areat position(s) 267, 283+287, 296-299, 303-304, 331-332, 339+342,347-349+351, 364-366, 408, 410, 416-418, 426-427+429-430, 432-434, 440,445+449, 449+453, 460+462, and/or 477 of the corresponding wild-typet-PA, where the "+" indicates replacements only at the positionsdesignated, and the "-" indicates replacements at all positionsdesignated.

For the alanine scanning mutagenesis, it is preferable that an aminoacid be substituted that will neutralize the charge of the correspondingamino acid of the wild-type t-PA, rather than confer an opposite chargeon the molecule. Any hydrophobic, essentially uncharged or oppositelycharged amino acid can be used, including, as preferred, alanine,glycine, serine, threonine, asparagine, glutamine, valine, leucine,isoleucine, phenylalanine, or tyrosine. Among these, small amino acids,such as alanine, serine and threonine, are preferred over larger aminoacids such as valine, leucine, and isoleucine. Charged amino acids suchas aspartic acid or glutamic acid are less preferred.

More preferably, the amino acid used for replacement is either alanine,serine, threonine, asparagine, glutamine, phenylalanine, or tyrosine,and more preferably still, alanine, serine, or threonine. Alanine is themost preferred amino acid for this purpose because it eliminates theside-chain beyond the beta-carbon and is less likely to alter themain-chain conformation of the wild-type t-PA molecule. Further, alanineis frequently found in both buried and exposed positions (Creighton, T.E., in The Proteins (eds. W. H. Freeman & Co., N.Y.); Chothia, C. (1976)J. Mol. Biol., 150: 1).

Preferred variants of this latter class of zymogens or fibrin (or plasmaclot) specific variants are those that are inclusive of modificationswithin the protease domain of t-PA with the exception of the variantsD365A t-PA, R462L t-PA, A473S t-PA, d296-304 t-PA, d296-302 t-PA, R298Et-PA, R299E t-PA, R304E t-PA, R304S t-PA, d296-299 t-PA, and K296E,R298E, R299E t-PA.

More specifically, they are those that have one or more substitutions atposition(s) 267, 283, 287, 296, 297, 298, 299, 303, 304, 331, 332, 339,342, 347, 348, 349, 351, 364, 365, 366, 408, 410, 416, 417, 418, 426,427, 429, 430, 432, 434, 440, 445, 449, 453, 460, 462, or 477, orcombinations thereof, of the corresponding wild-type t-PA.

More preferably, the protease domain variants have substitutions atposition(s) 267, 283+287, 296-299, 303-304, 331-332, 339+342,347-349+351, 364-366, 408, 410, 416-418, 426-427+429-430, 432+434, 440,445+449, 449+453, 460+462, and 477 of the corresponding wild-type t-PA,where the "+" indicates alterations only at the positions designated,not positions in between, and the "-" indicates alterations at allpositions designated, including those in between.

Still more preferably, the protease domain variants of the latter classof zymogens or fibrin (or plasma clot) specific variants are R267A t-PA,D283A, H287A t-PA, K296A, H297A, R298A, R299A t-PA, E303A, R304A t-PA,H331A, H332A t-PA, R339A, R342A t-PA, E347A, E348A, E349A, K351A t-PA,D364A, D365A, D366A t-PA, E408A t-PA, E410A t-PA, K416A, H417A, E418At-PA, E426A, R427A, K429A, E430A t-PA, H432A, R434A t-PA, R440A t-PA,H445A, R449A t-PA, R449A, D453A t-PA, D460A, R462A t-PA, and D477A t-PA.

Of these, the most preferred substitution is an alanine residue in placeof each of the existing residues at 296-299 of wild-type t-PA, i.e.,K296A, H297A, R298A, R299A t-PA.

The preferred insertional variants that may exhibit zymogenic orfibrin-specific activity are those wherein one or more amino acids areinserted after the amino acids at positions 296, 297, 298, and/or 299.Also preferred for this purpose are those protease domain variants withan insertion consisting of either tyrosine, asparagine, lysine,arginine, or glutamine.

Other variants with one or more amino acid alterations (deletions,substitutions, or insertions, but preferably substitutions) within theprotease domain (amino acids 264-527) of the native t-PA molecule areidentifiable that exhibit zymogenic properties as compared to thewild-type t-PA, using one or more of the screening tests provided below.

The t-PA variants herein, in addition to being altered from the nativesequence at one or more protease domain sites so as to display zymogenicand/or fibrin (or plasma clot) specific properties, also optionallycontain substitutions, deletions, or insertions of residues in otherregions of the native sequence to improve certain properties of themolecule, provided that changes are not made that prevent the cleavageof the one-chain form of t-PA to its two-chain form or otherwise alter adesirable biological property conferred on the molecule by thealteration(s) in the protease domain of the present invention. Thepreferred alterations in these other domains are provided above in thelists of the most preferred zymogen variants of the first type.

For example, the variants herein are suitably devoid of at least aportion of the finger domain, the growth factor domain, and/or thekringle 1 domain, and/or devoid of glycosylation potential at theglycosylation site surrounding amino acid 184, and suitably containamino acid modifications in the putative lysine binding site of kringle1 or 2.

In addition, fibrin binding of t-PA can be modulated, most preferablyrestored or increased, by appropriate substitutions of positively ornegatively charged amino acid residues on the opposite edges of theputative ligand binding pocket of the kringle 2 domain of t-PA. Thevariants herein are generally prepared by site-directed mutagenesis orby excision/ligation techniques described further hereinbelow.

Specific examples of such variants include a molecule devoid of aminoacids 1 to 44 (designated d1-44) and a molecule having aspartic acid atposition 184 (designated N184D). Variants devoid of amino acids 1 to 44are described more fully in WO 89/00197, supra.

All of the above variants are optionally modified in various otherregions of the molecule, if such modifications still satisfy thecriteria expressed herein for zymogenic and/or fibrin (or plasma clot)specific characteristics. Such modifications include, for example:

1. Kringle 1 modifications, for example, deletion of about 92 to 179,and/or

2. Kringle 2 modifications, for example, deletion of about 174-261 ormodification in the region of amino acids about 205-215, especially210-213, and/or

3. Amino acids about 244-255, especially 252 or its site, and/or

4. Amino acids about 233-242, especially 236-238, and/or

5. Known glycosylation sites such as amino acid 184, and/or

6. Glycosylation within the growth factor domain, as described in U.S.Appln. Ser. No. 07/196,909 filed May 20, 1988, now abandoned, thedisclosure of which is incorporated herein by reference. Briefly, thet-PA molecule is N- or O-linked glycosylated within its growth factordomain, preferably at position 67-69, where the tyrosine at position 67is replaced with an asparagine residue, to alter the half-life of thet-PA molecule.

Many of these modifications may significantly alter clearance rates andfibrin binding relative to native t-PA. The practitioner skilled in theart will be able to determine by the appropriate assay what the optimumproperties of each variant are that are desired in any particularinstance.

The modification to change or insert the appropriate amino acid(s) inthe native molecule to effect the above sequence variations isaccomplished by any means known in the art, such as, e.g., site-directedmutagenesis or ligation of the appropriate sequence into the DNAencoding the relevant protein, as described below.

2. Site-Specific Mutagenesis

Preparation of t-PA variants in accordance herewith is preferablyachieved by site-specific mutagenesis of DNA that encodes an earlierprepared variant or a nonvariant version of the protein. Site-specificmutagenesis allows the production of t-PA variants through the use ofspecific oligonucleotide sequences that encode the DNA sequence of thedesired mutation, as well as a sufficient number of adjacent nucleotidesto provide a primer sequence of sufficient size and sequence complexityto form a stable duplex on both sides of the junction being traversed.Typically, a primer of about 20 to 25 nucleotides in length ispreferred, with about 5 to 10 residues on both sides of the junction ofthe sequence being altered. In general, the technique of site-specificmutagenesis is well known in the art as exemplified by publications suchas Adelman et al., DNA, 2: 183 (1983), the disclosure of which isincorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily commercially available and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol., 153: 3 (1987)) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant t-PA. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA), 75: 5765 (1978). This primer is thenannealed with the single-stranded t-PA sequence-containing vector, andsubjected to DNA-polymerizing enzymes such as E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatecells such as JM101 cells and clones are selected, via hybridization toa radioactive probe consisting of the ³² P-labeled mutagenesis primer,that include recombinant vectors bearing the mutated sequencearrangement.

After such a clone is selected, the mutated t-PA region may be removedand placed in an appropriate vector for t-PA production, generally anexpression vector of the type that typically is employed fortransformation of an appropriate eukaryotic host. In the context of thepresent invention, Chinese hamster ovary (CHO) cells or 293 (humankidney cells described by Graham et al., J. Gen. Virol., 36: 59 (1977))are preferred for the preparation of long-term stable t-PA producers.However, the invention is not limited to CHO production, as it is knownthat numerous other cell types are suitably employed, particularly whereone desires only transient production of the enzyme for test purposes.For example, described below is a transient system employing 293 cellsthat provides a convenient system for production of t-PA variants foranalytical purposes.

3. Cleavage/Ligation Technique

Another method for making mutations in the DNA sequence encoding thet-PA involves cleaving the DNA encoding the t-PA at the appropriateposition by digestion with restriction enzymes, recovering the properlycleaved DNA, synthesizing an oligonucleotide encoding the desired aminoacid and flanking regions such as polylinkers with blunt ends (or,instead of using polylinkers, digesting the synthetic oligonucleotidewith the restriction enzymes also used to cleave the t-PA-encoding DNA,thereby creating cohesive termini), and ligating the synthetic DNA intothe remainder of the t-PA-encoding structural gene.

4. Host Cell Cultures and Vectors

Although Chinese hamster ovary (CHO) expression ultimately is preferredfor t-PA production, the vectors and methods disclosed herein aresuitable for use in host cells over a wide range of eukaryoticorganisms.

In general, of course, prokaryotes are preferred for the initial cloningof DNA sequences and constructing the vectors useful in the invention.For example, E. coli K12 strain 294 (ATCC No. 31,446) and E. coli strainW3110 (ATCC No. 27,325) are particularly useful. Other suitablemicrobial strains include E. coli strains such as E. coli B, and E. coliX1776 (ATCC No. 31,537). These examples are, of course, intended to beillustrative rather than limiting.

Prokaryotes also are useful for expression. The aforementioned strains,as well as bacilli such as Bacillus subtilis, and otherenterobacteriaceae such as, e.g., Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species are examples of useful hostsfor expression.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences that are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., Gene, 2: 95 (1977)). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid or phage, must also contain, or be modified tocontain, promoters that can be used by the microbial organism forexpression of its own proteins.

Those promoters most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase) and lactose promoter systems(Chang et al., Nature, 375: 615 (1978); Itakura et al., Science, 198:1056 (1977); Goeddel et al., Nature, 281: 544 (1979)) and a tryptophan(trp) promoter system (Goeddel et al., Nucl. Acids Res., 8: 4057 (1980);EPO Appl. Publ. No. 36,776), and the alkaline phosphatase systems. Whilethese are the most commonly used, other microbial promoters have beendiscovered and utilized, and details concerning their nucleotidesequences have been published, enabling a skilled worker to ligate themfunctionally with plasmid vectors (see, e.g., Siebenlist et al., Cell,20: 269 (1980)).

In addition to prokaryotes, eukaryotic microbes, such as yeasts, alsoare suitably used herein. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among eukaryotic microorganisms,although a number of other strains are commonly available. For example,for expression in Saccharomyces, the plasmid YRp7 (Stinchcombet al.,Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemperet al., Gene, 10: 157 (1980)) is commonly used. This plasmid alreadycontains the trp1 gene that provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, for example,ATCC No. 44,076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)). The presenceof the trp1 lesion as a characteristic of the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073(1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.,7: 149 (1968); Holland et al., Biochemistry, 17: 4900 (1978)), such asenolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In the construction ofsuitable expression plasmids, the termination sequences associated withthese genes are also ligated into the expression vector 3' of thesequence desired to be expressed to provide polyadenylation of the mRNAand termination. Other promoters that have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years [Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)]. Examples of such useful host cell linesare VERO and HeLa cells, CHO cell lines, and W138, BHK, COS-7, 293, andMDCK cell lines. Expression vectors for such cells ordinarily include(if necessary) an origin of replication, a promoter located in front ofthe gene to be expressed, along with any necessary ribosome bindingsites, RNA splice sites, polyadenylation sites, and transcriptionalterminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment that also contains the SV40 viral origin of replication (Fierset al., Nature, 273: 113 (1978)). Smaller or larger SV40 fragments arealso suitably used, provided there is included the approximately 250-bpsequence extending from the HindIII site toward the BglI site located inthe viral origin of replication. Further, it is also possible, and oftendesirable, to utilize promoter or control sequences normally associatedwith the desired gene sequence, provided such control sequences arecompatible with the host cell systems.

An origin of replication typically is provided either by construction ofthe vector to include an exogenous origin, such as may be derived fromSV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or by thehost cell chromosomal replication mechanism. If the vector is integratedinto the host cell chromosome, the latter is often sufficient.

Satisfactory amounts of human t-PA are produced by cell cultures;however, refinements, using a secondary coding sequence, serve toenhance production levels even further. The secondary coding sequencecomprises dihydrofolate reductase (DHFR) that is affected by anexternally controlled parameter, such as methotrexate (MTX), thuspermitting control of expression by control of the MTX concentration.

In the selection of a preferred host cell for transfection by thevectors of the invention that comprise DNA sequences encoding bothvariant t-PA and DHFR protein, it is appropriate to consider the type ofDHFR protein employed. If wild-type DHFR protein is employed, it ispreferable to select a host cell that is deficient in DHFR, thuspermitting the use of the DHFR coding sequence as a marker forsuccessful transfection in selective medium that lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is the CHOcell line deficient in DHFR activity, prepared and propagated, asdescribed by Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77: 4216(1980).

On the other hand, if DHFR protein with low binding affinity for MTX isused as the controlling sequence, it is not necessary to useDHFR-deficient cells. Because the mutant DHFR is resistant to MTX,MTX-containing media can be used as a means of selection, provided thatthe host cells are themselves MTX sensitive. Most eukaryotic cells thatare capable of absorbing MTX appear to be sensitive to MTX. One suchuseful cell line is a CHO line, CHO-K1 (ATCC No. CCL 61).

5. Typical Cloning and Expression Methodology Employable

If mammalian cells are used as host cells, transfection generally iscarried out by the calcium phosphate precipitation method as describedby Graham and Van der Eb, Virology, 52: 546 (1978). However, othermethods for introducing DNA into cells such as nuclear injection,electroporation, or protoplast fusion are also suitably used.

If yeast are used as the host, transfection is generally accomplishedusing polyethylene glycol, as taught by Hinnen, Proc. Natl. Acad. Sci.U.S.A., 75: 1929-1933 (1978).

If prokaryotic cells or cells that contain substantial cell wallconstructions are used, the preferred method of transfection is calciumtreatment using calcium as described by Cohen et al., Proc. Natl. Acad.Sci. (USA) 69: 2110 (1972), or more recently electroporation.

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to form the plasmids required.

Cleavage is performed by treating with restriction enzyme (or enzymes)in suitable buffer. In general, about 1 μg plasmid or DNA fragments isused with about 1 unit of enzyme in about 20 μl of buffer solution.(Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by themanufacturer.) Incubation times of about onehour at 37° C. are workable. After incubation, protein is removed byextraction with phenol and chloroform, and the nucleic acid is recoveredfrom the aqueous fraction by precipitation with ethanol.

If blunt ends are required, the preparation may be treated for 15minutes at 15° C. with 10 units of the Klenow fragment of DNA PolymeraseI (Klenow), phenol-chloroform extracted, and ethanol precipitated.

Size separation of the cleaved fragments is performed using 6 percentpolyacrylamide gel described by Goeddel et al., Nucleic Acids Res., 8:4057 (1980).

For ligation, approximately equimolar amounts of the desired components,suitably end tailored to provide correct matching, are treated withabout 10 units T4 DNA ligase per 0.5 μg DNA. (When cleaved vectors areused as components, it may be useful to prevent religation of thecleaved vector by pretreatment with bacterial alkaline phosphatase.)

As discussed above, t-PA variants are preferably produced by means ofspecific mutation. Variants useful in the practice of the presentinvention are formed most readily through the use of specificoligonucleotide sequences that encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a sequence of sufficient size and sequence complexity to form astable duplex on both sides of the mutation being traversed.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are typically used to transform E. coli K12 strain 294(ATCC 31,446) or other suitable E. coli strains, and successfultransformants selected by ampicillin or tetracycline resistance whereappropriate. Plasmids from the transformants are prepared and analyzedby restriction mapping and/or DNA sequencing by the method of Messing etal., Nucleic Acids Res., 9: 309 (1981) or by the method of Maxam et al.,Methods of Enzymology, 65: 499 (1980) .

After introduction of the DNA into the mammalian cell host and selectionin medium for stable transformants, amplification of DHFR-protein-codingsequences is effected by growing host cell cultures in the presence ofapproximately 20,000-500,000 pM concentrations of MTX, a competitiveinhibitor of DHFR activity. The effective range of concentration ishighly dependent, of course, upon the nature of the DHFR gene andprotein and the characteristics of the host. Clearly, generally definedupper and lower limits cannot be ascertained. Suitable concentrations ofother folic acid analogs or other compounds that inhibit DHFR could alsobe used. MTX itself is, however, convenient, readily available, andeffective.

In order to simplify the examples certain frequently occurring methodswill be referenced by shorthand phrases.

"Plasmids" are designated by a low case p followed by an alphanumericdesignation. The starting plasmids herein are commercially available,are publicly available on an unrestricted basis, or can be constructedfrom such available plasmids in accord with published procedures. Inaddition, other equivalent plasmids are known in the art and will beapparent to the ordinary artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the sites for which each is specific iscalled a restriction site. The various restriction enzymes used hereinare commercially available and their reaction conditions, cofactors andother requirements as established by the enzyme suppliers were used.Restriction enzymes commonly are designated by abbreviations composed ofa capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1μg of plasmid or DNA fragment is used with about 2 units of enzyme inabout 20 μl of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation times of about 1 hour at 37° C. are ordinarilyused, but may vary in accordance with the supplier's instructions. Afterincubation, protein is removed by extraction with phenol and chloroform,and the digested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5' phosphates to prevent the two restriction cleavedends of a DNA fragment from "circularizing" or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5' terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional (T. Maniatis et al., 1982, MolecularCloning pp. 133-134).

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see R. Lawn et al., 1981, Nuclei Acids Res. 9:6103-6114, and D. Goeddel et al., 1980, Nucleic Acids Res. 8: 4057.

"Southern Analysis" is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labelled oligonucleotide or DNA fragment. For the purposesherein, unless otherwise provided, Southern analysis shall meanseparation of digests on 1 percent agarose, denaturation and transfer tonitrocellulose by the method of E. Southern, 1975, J. Mol. Biol. 98:503-517, and hybridization as described by T. Maniatis et al., 1978,Cell 15: 687-701.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or chromosomalintegrant. Unless otherwise provided, the method used herein fortransformation of E. coli is the CaCl₂ method of Mandel et al., 1970, J.Mol. Biol. 53: 154.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (T. Maniatis et al., Id., p.146). Unless otherwise provided, ligation may be accomplished usingknownbuffers and conditions with 10 units of T4 DNA ligase ("ligase")per 0.5 mg of approximately equimolar amounts of the DNA fragments to beligated.

"Preparation" of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofManiatis et al., Id., p. 90, may be used.

"Oligonucleotides" are short length single or double strandedpolydeoxynucleotides that are chemically synthesized by known methodsand then purified on polyacrylamide gels.

C. Pharmaceutical Compositions

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe t-PA product hereof is combined in admixture with a pharmaceuticallyacceptable carrier vehicle. Suitable carrier vehicles and theirformulation, inclusive of other human proteins, e.g., human serumalbumin, are described, for example, in Remington's PharmaceuticalSciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al.,the disclosure of which is hereby incorporated by reference. Suchcompositions will typically contain an effective amount of the variantherein, for example, from about 0.5 to about 5 mg/ml, together with asuitable amount of carrier vehicle to prepare pharmaceuticallyacceptable compositions suitable for effective administration to thehost. The t-PA variant herein may be administered parenterally tosubjects suffering from cardiovascular diseases or conditions, or byother methods that ensure its delivery to the bloodstream in aneffective form.

Compositions particularly well suited for the clinical administration ofvariant t-PA products employed in the practice of the present inventioninclude, for example, sterile aqueous solutions, or sterile hydratablepowders such as lyophilized protein. It is generally desirable toinclude further in the formulation an appropriate amount of apharmaceutically acceptable salt, generally in an amount sufficient torender the formulation isotonic. A pH regulator such as arginine base,and phosphoric acid, are also typically included in sufficientquantities to maintain an appropriate pH, generally from 5.5 to 7.5.Moreover, for improvement of shelf-life or stability of aqueousformulations, it may also be desirable to include further agents such asglycerol. In this manner, variant t-PA formulations are renderedappropriate for parenteral administration, and, in particular,intravenous administration.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. For example, in the treatment of deep vein thrombosis orperipheral vascular disease, "bolus" doses, on the order of about 0.05to about 0.2 mg/kg, will typically be preferred with subsequentadministrations, on the order of about 0.1 to about 0.2 mg/kg, beinggiven to maintain an approximately constant blood level, preferably onthe order of about 3 mg/ml.

However, for use in connection with emergency medical care facilitieswhere infusion capability is generally not available and due to thegenerally critical nature of the underlying disease (e.g., embolism,infarct), it will generally be desirable to provide somewhat largerinitial doses, such as an intravenous bolus on the order of about 0.3mg/kg.

For example, the t-PA variant hereof is suitably administeredparenterally to subjects suffering from cardiovascular diseases orconditions. Dosage and dose rate may be parallel to or higher than thatcurrently in use in clinical investigations of other cardiovascular,thrombolytic agents, e.g., about 1-2 mg/kg body weight as an intravenousor intra-arterial dose over 1.5 to 12 hours in human patients sufferingfrom myocardial infarction, pulmonary embolism, etc. Higher doses may betolerated because the variants herein have lower side effects thanwild-type t-PA, leading to faster and more complete clot lysis.

As one example of an appropriate dosage form, a vial containing 50 mgt-PA, arginine, phosphoric acid, and polysorbate 80 is reconstitutedwith 50 ml sterile water for injection and mixed with a suitable volumeof 0.9 percent sodium chloride injection.

The t-PA variants herein also are useful to prevent fibrin deposition oradhesion formation or reformation. One embodiment of this use isdescribed in U.S. Ser. No. 07/125,319 filed Nov. 25, 1987, thedisclosure of which is incorporated herein by reference. Generally, suchtreatment involves topical administration of a composition to a site ofpotential fibrin or adhesion formation wherein the composition comprisesa therapeutically effective amount of the t-PA variant in a sparinglysoluble form that is continuously released at that site for a period oftime of about from three days to two weeks. Typically, the t-PA variantis administered at a dosage sufficient to prevent fibrin deposition orformation of adhesions following surgery, infection, trauma, orinflammation. Typically, this amount is from 0.02 mg/g of gel to 25 mg/gof gel, with preferred amounts from 0.20 mg/g gel to about 2.5 mg/g ofgel, most preferably from 0.25 mg/g to about 1.0 mg/g of gel.

The vehicle in which the t-PA is typically formulated for preventingadhesion formation in a semisolid, mucilaginous pharmaceutically inertcarrier for positioning the enzyme at the site of potential adhesionformation. Such a carrier includes long-chain hydrocarbons or vegetableoils and waxes composed of mixtures of saturated and unsaturated fattyacid glycerides or mixtures of modified saturated and unsaturated fattyacid glycerides. Examples include semisolid vehicles such as petroleumjelly or semi-synthetic glycerides, polyhydroxy solvents such asglycerol, long-chain hydrocarbons, bioerodable polymers, or liposomes.

The following examples are intended merely to illustrate the best modenow known for practicing the invention, but the invention is not to beconsidered limited thereto.

All literature and patent application citations herein are expresslyincorporated by reference.

EXAMPLE I A. Preparation and Utilization of Expression Vector forRecombinant Production of the t-PA Variants Hereof

1. Construction of Plasmid p7-1H

a) Plasmid pCISt-PA

Plasmid pCISt-PA was prepared as follows. The vector pCIHt-PA containingthe cytomegalovirus enhancer and promoter, the cytomegalovirus splicedonor site and intron, the Ig variable region splice acceptor site, thecDNA-encoding t-PA (Pennica et al., Nature, 301: 214 (1983)) and thehepatitis surface antigen polyadenylation and transcription terminationsite was constructed first:

The vector pF8CIS containing the cytomegalovirus enhancer (Boshart etal., Cell, 41: 520 (1985)) and promoter (Thomsen et al., Proc. Natl.Acad. Sci. (U.S.A.) 81: 659 (1984)), the cytomegalovirus splice donorsite and a portion of an intron (Sternberg et al., J. of Virol., 49: 190(1984)), the Ig variable region intron and splice acceptor site, thecDNA encoding factor VIII, and the SV40 polyadenylation site wasconstructed. The three parts of the construction are detailed below.

1. The ampicillin resistance marker and replication origin of the finalvector was derived from the starting plasmid pUC13pML, a variant of theplasmid pML (Lusky et al., Nature, 293: 79 (1981)). pUC13pML wasconstructed by transferring the polylinker of pUC13 (Veira et al., Gene,19: 259 (1982)) to the EcoRI and HindIII sites of pML. A second startingplasmid pUC8CMV was the source of the CMV enhancer, promoter and splicedonor sequence. pUC8CMV was constructed by inserting nucleotides 1through 732 for the CMV enhancer, promoter and splice donor sequenceinto the blunted PstI and SphI sites of pUC8--Veira et al., supra.Synthetic BamHI-HindIII linkers (commercially available from New EnglandBiolabs) were ligated to the cohesive BamHI end, creating a HindIIIsite. Following this ligation a HindIII-HincII digest was performed.This digest yielded a fragment of approximately 800 bp that containedthe CMV enhancer, promoter and splice donor site. Following gelisolation, this 800-bp fragment was ligated to a 2900-bp piece ofpUC13pML. The fragment required for the construction of pF8CIS wasobtained by digestion of the above intermediate plasmid with SalI andHindIII. This 3123-bp piece contained the resistance marker forampicillin, the origin of replication from pUC13pML, and the controlsequences for the CMV, including the enhancer, promoter, and splicedonor site.

2. The Ig variable region intron and splice acceptor sequence wasconstructed using a synthetic oligomer. A 99-mer and a 30-mer werechemically synthesized having the following sequence for the IgG intronand splice acceptor site (Bothwell et al., Cell, 24:625 (1981)):

    ______________________________________                                         1  5'-AGTAGCAAGCTTGACGTGTGGCAGGCTTGA . . .                                   31  GATCTGGCCATACACTTGAGTGACAATGA . . .                                       60  CATCCACTTTGCCTTTCTCTCCACAGGT . . .                                        88  GTCCACTCCCAG-3'                                                            1  3'-CAGGTGAGGGTGCAGCTTGACGTCGTCGGA-5'                                      ______________________________________                                    

DNA polymerase I (Klenow fragment) filled in the synthetic piece andcreated a double-stranded fragment (Wartell et al., Gene, 9: 307(1980)). This was followed by a double digest of PstI and HindIII. Thissynthetic linker was cloned into pUC13 (Veira et al., Supra) at the PstIand HindIII sites. The clone containing the synthetic oligonucleotide,labeled pUCIg.10, was digested with PstI. A ClaI site was added to thisfragment by use of a PstI-ClaI linker. Following digestion with HindIIIa 118- bp piece containing part of the Ig intron and the Ig variableregion splice acceptor was gel isolated.

3. The third part of the construction scheme replaced the hepatitissurface antigen 3' end with the polyadenylation site and transcriptiontermination site of the early region of SV40. A vector, pUC.SV40,containing the SV40 sequences was inserted into pUC8 at the BamHI sitedescribed in Veira et al., supra. pUC.SV40 was then digested with EcoRIand HpaI. A 143-bp fragment containing only the SV40 polyadenylationsite was gel isolated from this digest. Two additional fragments weregel isolated following digestion of pSVE.8c1D (EPO Pub. No. 160,457).The 4.8-kb fragment generated by EcoRI and ClaI digest contains theSV40-DHFR transcription unit, the origin of replication of pML, and theampicillin resistance marker. The 7.5-kb fragment produced followingdigestion with ClaI and HpaI contains the cDNA for Factor VIII. Athree-part ligation yields pSVE.8c24D. This intermediate plasmid wasdigested by ClaI and SalI to give a 9611-bp fragment containing the cDNAfor Factor VIII with the SV40 polyadenylation and transcriptiontermination sites followed by the SV40 DHFR transcription unit.

The final three-part ligation to yield pF8CIS used: a) the 3123-bpSalI-HindIII fragment containing the origin of replication, theampicillin resistance marker and the CMV enhancer, promoter and splicedonor; b) the 118-bp HindIII-ClaI fragment containing the Ig intron andsplice acceptor; and c) a 9611-bp ClaI-SalI fragment containing the cDNAfor Factor VIII, SV40 polyadenylation site, and the SV40 DHFRtranscription unit.

Next, the completion of the construction of plasmid pCIHt-PA fromintermediate plasmid pCla t-PA and plasmid pF8CIS (above) wasundertaken:

The t-PA cDNA was first cloned into pML to provide a ClaI site at the 5'end of the gene. To do this, a 3238-bp HindIII fragment from pSVpa-DHFR(otherwise referred to as pETPFR, supra) was inserted into the HindIIIsite of pML (Lusky et al., supra). Colonies were screened for clonesthat have the 5' end of the cDNA juxtaposed to the ClaI site. Theintermediate plasmid was labeled pCLAt-PA. A t-PA cDNA followed by the3'-polyadenylation regions was isolated as a ClaI-KpnI fragment of 2870bp. This fragment was ligated to the 5146-bp fragment of pF8CIS. ThisClaI-KpnI fragment of the CIS vector provided the 5' control region, aSV40-DHFR transcriptional unit, the ampicillin resistance gene, and theorigin region from pML. See FIG. 2.

Expression levels of t-PA were obtained by transfecting CHO or 293 cellswith pCIHt-PA, in accordance with methods generally known per se anddescribed supra. Media from the transfected 293 cells, for example, wereassayed, demonstrating that pCIHt-PA produced 420 ng/ml of t-PA.

The vector pCISt-PA containing the cytomegalovirus enhancer andpromoter, the cytomegalovirus splice donor site and intron, the Igvariable region splice acceptor site, the cDNA encoding t-PA, and thepSV40 polyadenylation sequence was finally constructed as follows:

The starting vectors for this construction were pCIHt-PA and pF8CIS(supra). The latter vector has the same 5' controls as pCIHt-PA, butincludes the cDNA for Factor VIII and the SV40 polyadenylation site.SacII was used to cleave 3' of the t-PA cDNA. The resultant 3' overhangwas blunted by T4 polymerase. pCIHt-PA was then cut with ClaI. This siteseparates the chimeric intron cleaving between the CMV intronicsequences and the Ig variable region intron. An 2870-bp fragment was gelisolated from the ClaI treatment. The SV40 polyadenylation site, DHFR,transcription control, bacterial origin of replication, and amp^(r)gene, as well as the CMV enhancer and promoter and splice donor wereisolated from pF8CIS. These elements were isolated into fragments as a2525-bp Sal-BamHI fragment and a HpaI-Sal and 3113-bp fragment. Athree-part ligation of the KpnI (blunt)-ClaI fragment with the HpaI-Salfragment and Sal to BamHI fragment yields pCISt-PA, which was expressedin both CHO and 293 cells as discussed above for plasmid pCIHt-PA,giving 55 and 3000 ng/ml of t-PA, respectively. See FIG. 3.

b) Final Construction of p7-1H

The plasmid pCISt-PA was digested with SpeI, then treated with E. coliDNA polymerase I large fragment (Klenow) and deoxyribonucleosidetriphosphates to create blunt ends. The resulting linear fragment wasligated, using T4 DNA ligase, to the 0.45kb-RsaI/AhaIII fragmentcontaining the + strand origin from the single-stranded DNA phage, f1,as described in Zinder et al., Microbiol. Rev., 49: 101 (1985). Ligationproducts were isolated with the f1 origin inserted in both possibleorientations at the SpeI site of the pCISt-PA fragment. A plasmidcontaining this origin, in such an orientation that the anti-sensestrand of the t-PA gene was packaged into virions in the presence ofhelper phage, was chosen and termed p7-1H. See FIG. 4.

2. Mutagenesis of Expression Plasmid

a) Template Preparation

Plasmid p7-1H was introduced into E. coli strain JM101 (ATCC No. 33,876)via CaCl₂ -mediated transformation. These cells were then infected withthe helper virus M13K07 and single-stranded p7-1H DNA was prepared asdescribed by Veira et al., Meth. Enzymol., 153: 3 (1987). Briefly, to0.3 ml of a saturated culture of transformed cells in 2YT broth wasadded 10⁹ -10¹⁰ pfu of M13K07 and the mixture was incubated for 15 min.at 37° C. 1.5 ml of fresh 2YT broth, containing 50 μg/ml carbenicillin,was added and the culture was gently shaken for 16 hours at 37° C. Afterthe cells were pelleted, phage and packaged plasmid DNA were harvested,and single-stranded DNA was prepared as described by Anderson, Nucl.Acids. Res., 9: 3015 (1981).

b) Site-directed in vitro Mutagenesis

Mutagenesis on p7-1H was carried out using the oligodeoxyribonucleotide,5'-CGGAGAGCGGCACCTGTGCGGGG-3', essentially as described by Zoller etal., Meth. Enzymol., 100: 468 (1983), except that the mutant, with themutation phe305→his305, was identified by colony hybridization ratherthan plaque hybridization. Mutations were verified by DNA sequencingdirectly on the single-stranded plasmid DNA using the dideoxynucleotidechain termination method (Sanger et al., Proc. Natl. Acad. Sci. (U.S.A.)74: 5463 (1977)).

3. Expression and Purification

a) Plasmid Preparation

Transformed cells were grown to saturation in 500-ml LB broth containing50 μg/ml carbenicillin. Cells were pelleted by centrifugation andresuspended in 40 ml of 50 mM glucose, 10 mM EDTA, 25 mMTris-HCl (pH8.0). To this suspension was added 60 ml of 1% sodium dodecyl sulfate,0.07M NaOH, and the mixture was incubated for 2 min at 25° C., then at10 min. at 0° C. To this 52 ml of 4M acetic acid, 3M sodium acetate wasadded and the mixture was incubated for 30 min. at 0° C. This was thencentrifuged at 11,500 rpm for 20 min., the supernatant mixed with twovolumes of 100% cold ethanol, and the resulting precipitate harvested bycentrifugation. The pellet, containing plasmid DNA and RNA, was driedand redissolved in 100 mM Tris (pH 8.0), 10 mM EDTA, 1 μg/ml RNase A.After the resulting solution was clarified by centrifugation, it wasadjusted to 0.5 mg/ml in ethidium bromide and an equal weight of CsClwas added. The DNA was then centrifuged in a Beckman VTI65 rotor for 16hours at 55,000 rpm at 18° C. The DNA band was harvested by sidepuncture, extracted with n-butanol to remove the ethidium bromide,diluted with H₂ O, and precipitated by ethanol. DNA was redissolved in10 mM Tris (pH 8.0), 1 mM EDTA, to a final concentration of 1 mg/ml.

b) Transfection and Expression

293 cells were grown to confluence. Ten μg of t-PA plasmid DNA mutantwas mixed with 1 μg of DNA encoding the VA RNA gene (Thimmappaya et al.,Cell, 31: 543 (1982)) and dissolved in 500 μl of 1 mM Tris--HCl, 0.1 mMEDTA, 0.227M CaCl₂. Added to this (dropwise while vortexing) was 500 μlof 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NAPO₄, and the precipitatewas allowed to form for 10 min. at 25° C. The suspended precipitate wasthen added to the cells (in 100 mM plate) and allowed to settle for fourhours in the incubator. The medium was then aspirated off and 2 ml of20% glycerol in phosphate-buffered saline (PBS) was added for 30 sec.The cells were washed twice with 5 ml of serum-free medium, then freshmedium was added and the cells were incubated for five days.

For the creation of stable CHO cell lines expressing the t-PA variant,the 1.4 kb BglII/ApaI fragment containing the bulk of the t-PA codingsequences (the BglII site spans codons -1 to 1 of full-lengtht-PA-encoding DNA and the ApaI site spans codons 465 to 466 offull-length t-PA-encoding DNA) may be ligated to the 6.0-kb BglII/ApaIfragment from the vector pPADHFR-6 (described in EPO Pat. Publn. No.93,619). The resultant plasmid is then introduced into CHO cells andinduced to over-express the t-PA variants by amplifying the codingsequence by means of selection in methotrexate-containing media.

c) Purification

Purification of the t-PA product was accomplished by passing theconditioned medium over a column (1-ml bed volume) of controlled glassbeads to which an anti-t-PA goat polyclonal A6 antibody (preparedaccording to standard methods known per se) had been coupled. Before themedium was loaded, the column was equilibrated with PBS and, afterloading, the column was equilibrated with 0.1M Tris-HCl (pH 7.5), 1MNaCl. The t-PA was eluted with 0.1M acetic acid, 0.15M NaCl, 0.02Marginine, 0.01% Tween 80 (pH 2.0), and fractions were immediatelyneutralized with Tris-base. Fractions were adjusted to 0.01% Tween 80before pooling. The t-PA was found on a reducing SDS gel to bepredominantly (80%) single chain.

B. Biological Assays

1. t-PA Quantitation

Protein concentrations were routinely determined by an ELISAstandardized to native-sequence t-PA (See EPO Pat. Publ. 93,619, supra).Protein purity and homogeneity were analyzed by polyacrylamide gelelectrophoresis in the presence of sodium dodecyl sulfate (PAGE-SDS)with the buffer system of Laemmli, Nature, 227: 680 (1970). Typically, 7to 17% gradient gels were used and proteins were visualized with thesilver-staining technique of Morrissey, Anal. Biochem., 117: 307 (1981).The t-PA variant prepared as described above was found to be pure andhomogeneous by this method.

2. S-2251 Assay

Results for clot lysis and S-2251 assays show averages of severalindependent observations (clot lysis, two determinations; S-2251, threedeterminations).

The ability of t-PA to activate plasminogen can be measured in an invitro assay by preincubating t-PA and plasminogen and then adding theplasmin-specific substrate H-D-valyl-H-leucyl-H-lysine-paranitroanilide(S-2251). The maximum rate of this reaction is observed in the presenceof fibrin(ogen) or fragments of fibrin(ogen) that act as stimulators ofthe reaction.

The plasmin-specific substrate S-2251 was used in a two-stage assay tomeasure the ability of the sample to activate plasminogen. Fibrinogencould be used as a stimulator by incubating the sample with 0.02 ml of a20 mg/ml fibrinogen solution in a total volume of 0.12 ml of 0.05MTris--HCl, 0.12M NaCl, 0.01% Tween 80, pH 7.4.

Glu-plasminogen solution (commercially available), 0.03 ml of a 2.0mg/ml solution in 0.05M Tris, 0.12M NaCl buffer, pH 8, was then added.After ten min. at 37° C., 0.35 ml of 0.86 mM S-2251 in 0.037M Tris,0.086 NaCl, 0.007% Tween 80, pH 7.4 was added. This mixture wasincubated for five minutes; then the reaction was stopped by theaddition of 0.1 ml of 50% glacial acetic acid. Absorbance at 405 nm wasmeasured. The activity was expressed as the change in absorbance pernanogram per minute in the presence of substrate.

The results are that the F305H variant, with fibrinogen, has 78% of thewild-type specific activity, which may be due to the lag before the A405increases.

3. Clot Lysis

Wild-type and F305H t-PA were assayed for their ability to lyse fibrinin the presence of saturating concentrations of plasminogen, accordingto the method of Carlsen et al., Anal. Biochem., 168: 428-435 (1988).The in vitro clot lysis assay measures the activity of t-PAs byturbidimetry using a microcentrifugal analyzer. A mixture of thrombinand t-PA test samples is centrifuged into a mixture of fibrinogen andplasminogen to initiate clot formation and subsequent clot dissolution.The resultant profile of absorbance versus time is analyzed to determinethe assay endpoint. Activities of the t-PA variants were compared to astandard curve of rt-PA (EPO Publ. No. 93,619, supra). The buffer usedthroughout the assay was 0.06M sodium phosphate, pH 7.4, containing0.01% (v/v) Tween 80 and 0.01% (w/v) sodium azide. Human thrombin was ata concentration of 33 units/ml. Fibrinogen (at 2.0 mg/ml clottableprotein) was chilled on wet ice to precipitate fibronectin and thengravity filtered. Glu-plasminogen was at a concentration of 1 mg/ml. Theanalyzer chamber temperature is set at 37° C. The loader is set todispense 20 μl of rt-PA (about 62.5 ng/ml to 1.0 μg/ml) as the samplefor the standard curve, or 20 μl of variant rtoPA at a concentration tocause lysis within the range of the standard curve. Twenty μl ofthrombin was used as the secondary reagent, and 200 μl of a 50:1 (v/v)fibrinogen: plasminogen mixture as the primary reagent. Theabsorbance/time program was used with a five-minute incubation time,340-nm filter, and 90-interval readings.

The results indicate that the F305H variant, using this assay, has about46% of the clot lysis activity of normal wild-type t-PA.

4. Fibrin Binding

The method for fibrin binding is a modification of the method describedby Rijken et al., J. Biol. Chem., 257: 2920 (1982). The t-PA sample tobe tested is added to a solution containing 0.05M Tris (pH 7.4), 0.12MNaCl, 0.01% Tween 80, 1 mg/ml human serum albumin, and variousconcentrations of plasminogen-free fibrin (0, 0.05, 0.1, 0.25, and 0.5mg/ml). The final volume of the reaction mixture was 1 ml, and the t-PAconcentration was 10 ng/ml for each sample. The samples were incubatedat 37° C. for 5 min., followed by the addition of 1 unit of thrombin.The samples were then incubated for one hour at 37° C. The clot wasremoved by centrifugation, and the amount of t-PA remaining unbound inthe supernatant was determined by ELISA.

The results (FIG. 5) show that predominantly single-chain F305H t-PA(closed triangles) binds to fibrin under the assay conditions employedalmost as well as one-chain wild-type t-PA (closed squares) and themixture of one-chain and two-chain wild-type t-PA (open triangles).Also, two-chain F305H t-PA (closed circles) binds fibrin at least aswell as two-chain wild-type t-PA (closed diamonds).

5. Zymogenic Kinetics by S-2251

a) Preparation of Fibrinogen

Human fibrinogen (Calbiochem) was made plasminogen free by applying itto a lysine-Sepharose column and collecting the flow-through. Theresulting fibrinogen pool was degraded by treatment withplasmin-Sepharose at room temperature overnight. The resultingclottability was 7%. The concentration was then adjusted to 1.51 mg/ml.

b) Procedure

The kinetics of the conversion of plasminogen to plasmin by thewild-type t-PA and the F305H t-PA were determined using the chromogenicplasmin substrate S-2251 in the presence of fibrinogen. The wild-typeand F305H t-PA molecules were used both in the predominantly one-chainform (obtained by purification as described previously) and in thetwo-chain, clipped form (obtained by incubation of the predominantlyone-chain form with plasmin-Sepharose for one hour at 37° C.).

In the presence of 1.2 μM plasmin-degraded fibrinogen fragments preparedas described above, the reactions were carried out at plasminogenconcentrations from 0.08 to 0.89 μM and t-PA concentrations of 2.3 to9.0 nM in 0.12M NaCl, 0.05M Tris, 0.01% Tween 80, pH 7.4. Theplasminogen, fibrinogen, and buffer were pre-incubated for three hoursat room temperature. The S-2251 was added for a final concentration of0.9 mM, and the samples were warmed to 37° C. for about 5 minutes. Attime zero, the t-PA samples were added, and the absorbance of eachsample was read in intervals of 30 seconds for ten minutes at 37° C.

The results are shown in FIGS. 6 and 7 (for wild-type, one-chain andtwo-chain t-PA, respectively) and in FIGS. 8 and 9 (for F305H, one-chainand two-chain t-PA, respectively). In the figures, the ordinate is theabsorbance at 405 nm and the abscissa is the square of the number ofminutes at which the absorbances were taken. The closed circlesrepresent 0.09 μM of plasminogen (and 15.7 nM wild-type two-chain t-PA,10.8 nM F305H two-chain t-PA, 16.1 nM wild-type one-chain t-PA, and 17.2nM F305H one-chain t-PA), the closed triangles represent 0.11 μM ofplasminogen (and 11.8 nM wild-type two-chain t-PA, 8.1 nM F305Htwo-chain t-PA, 12.1 nM wild-type one-chain t-PA, 12.9 nM F305Hone-chain t-PA), the closed squares represent 0.16 μM of plasminogen(and 9.8 nM wild-type two-chain t-PA, 6.7 nM F305H two-chain t-PA, 10.1nM wild-type one-chain t-PA, 10.8 nM F305H one-chain t-PA), the opencircles represent 0.22 μM of plasminogen (and 7.9 nM wild-type two-chaint-PA, 5.4 nM F305H two-chain t-PA, 8.1 nM wild-type one-chain t-PA, 8.6nM F305H one-chain t-PA), the open triangles represent 0.44 μM ofplasminogen (and 3.9 nM wild-type two-chain t-PA, 2.7 nM F305H two-chaint-PA, 4.0 nM wild-type one-chain t-PA, 4.3 nM F305H one-chain t-PA), andthe open squares represent 0.9 μM of plasminogen (and 3.9 nM wild-typetwo-chain t-PA, 2.7 nM F305H two-chain t-PA, 4.0 nM wild-type one-chaint-PA, 4.3 nM F305H one-chain t-PA).

The graphs show that only with the predominantly one-chain F305H variantdoes the absorbance exhibit a pronounced lag at early times in thereactions, and a rise in activity thereafter. Two-chain F305H variantdoes not exhibit this lag, but rather appears to have kinetic propertiessimilar to one- or two-chain wild-type t-PA. This behavior demonstratesthe zymogenic nature of the F305H variant that is not observed with thewild-type t-PA.

EXAMPLE II

A strategy known as alanine-scanning mutagenesis (ALA-scan), describedin Cunningham and Wells, supra, was employed for generation of the t-PAvariants evaluated in this example. This method involved theidentification of small surface regions of the t-PA protease domain thatcontain charged amino acid side chains. Without limitation to any onetheory, it is believed that either these regions containing clusters ofcharge, or neighboring regions, or both, are responsible for theinteraction of the t-PA molecule with its substrate and various othercompounds that may modulate its activity. The charged amino acids ineach region (i.e., Arg, Asp, His, Lys, and Glu) were replaced (oneregion per mutant molecule) with alanine to assess the importance of theparticular region to the overall activity of the t-PA molecule. Theresults are indicated below.

1. Construction of pRK7-t-PA

Plasmid pRK7 was used as the vector for generation of the t-PA mutants.pRK7 is identical to pRK5 (EP Pub. No. 307,247 published Mar. 15, 1989),except that the order of the endonuclease restriction sites in thepolylinker region between ClaI and HindIII is reversed. The t-PA cDNA(Pennica et al., Nature, 301: 214 (1983)) was prepared for insertioninto the vector by cutting with restriction endonuclease HindIII (whichcuts 49 base pairs 5' of the ATG start codon) and restrictionendonuclease BalI (which cuts 276 base pairs downstream of the TGA stopcodon). This cDNA was ligated into pRK7 previously cut with HindIII andSmaI using standard ligation methodology (Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,1982). This construct was named pRK7-t-PA.

2. Site-Directed Mutagenesis of pRK7-t-PA

Site-directed mutagenesis of t-PA cDNA was performed by the method ofTaylor et al., Nucl. Acids. Res., 13: 8765 (1985) using a kit purchasedfrom the Amersham Corporation (catalog number RPN 1253). For generationof the desired mutants, oligonucleotides of sequences coding for thedesired amino acid substitutions were synthesized and used as primers.These oligonucleotides were annealed to single-stranded pRK7-t-PA thathad been prepared by standard procedures (Viera et al., Meth. Enz., 143:3 (1987)).

A mixture of three deoxyribonucleotide triphosphates, deoxyriboadenosinetriphosphate (dATP) , deoxyriboguanosine triphosphate (dGTP) , anddeoxyribothymidine triphosphate (dTTP), was combined with a modifiedthiodeoxyribocytosine called dCTP (αS) provided in the kit by themanufacturer of the kit, and added to the single-stranded pRK7-t-PA towhich was annealed the oligonucleotide.

Upon addition of DNA polymerase to this mixture, a strand of DNAidentical to pRK7-t-PA except for the mutated bases was generated. Inaddition, this new strand of DNA contained dCTP (αS) instead of dCTP,which served to protect it from restriction endonuclease digestion.After the template strand of the double-stranded heteroduplex was nickedwith an appropriate restriction enzyme, the template strand was digestedwith ExoIII nuclease past the region that contained the mutagenicoligomer. The reaction was then stopped to leave a molecule that wasonly partly single-stranded. A complete double-stranded DNA homoduplexmolecule was then formed by DNA polymerase in the presence of all fourdeoxyribonucleotide triphosphates, ATP, and DNA ligase.

The following oligonucleotides were prepared to use as primers togenerate pRK7-t-PA molecules using the ALA-scan methodology describedabove:

5'-GGCTGTACTGGGCCAGGCCGCA-3' (R267A)

5'-GGCAGCCTGCCAGGGGGCGGAGGCGATGGCGGCGAAGAG-3' (D283A, H287A)

5'-CCGCTCTCCGGGCGACGCCGCGGCCGCGGCAAAGAT-3' (K296A, H297A, R298A, R299A)

5'-GCCCCCGCACAGGAACGCCGCTCCGGGCGA-3' (E303A, R304A)

5'-CTCCTGGAAGCAGGCGGCGGCAGA-3' (H322A)

5'-GTGGTGGGGCGGAAACGCCGCCTGGAACGA-3' (E326A, R327A)

5'-CAAGATCACCGTCAGGGCGGCGGGCGGAAA-3' (H331A, H332A)

5'-CTCGCCAGGGACCACCGCGTATGTTGCGCCCAAGAT-3' (R339A, R342A)

5'-TTTTTCGACTTCAAATGCCTGCGCCGCCGCGCCAGGGAC-3' (E347A, E348A, E349A,K351A)

5'-CTTATGGACAATGTATGCTGCGACTGCAAATTTCTG-3' (E353A, E355A, K356A)

5'-AGTGTCATCATCGAATGCCGCAGCGACAATGTA-3' (H360A, K361A, E362A)

5'-GTCATTGTCGTAAGTGGCAGCAGCGAATTCCTT-3∝ (D364A, D365A, D366A)

5'-CTGCAGCAGCGCAATGGCATTGGCGTAAGTGTC-3' (D369A, D371A)

5'-CTCCTGGGCACAGGCGGACGAAGCCGATGCCAGCTGCAG-3' (K378A, D380A, R383A)

5'-AAGGCACACAGTGGCGACCACGCTGCTCGCCTGGGCACA-3' (E387A, R392A)

5'-ACACTCCGTCCAGGCCGGCAGCTGCAGGGCCGCCGGGGG-3' (D400A, D405A)

5'-GGAGAGCTCACAGGCCGTCCAGTC-3' (E408A)

5'-GTAGCCGGAGAGGGCACACTCCGT-3' (E410A)

5'-AGGAGACAAGGCCGCAGCCGCGCCGTAGCC-3' ((K416A, H417A, E418A)

5'-TCTGACATGAGCCGCCGCCAGCGCCGCCGAATAGAA-3' (E426A, R427A, K429A, E430A)

5'-GGATGGGTACAGTGCGACAGCAGCCTCCTT-3' (H432A, R434A)

5'-TTGTGATGTGCAGGCGCTGGATGG-3' (R440A)

5'-GTCGGTGACTGTTGCGTTAAGTAAAGCTTGTGATGT-3' (H445A, R449A)

5'-ACACAGCATGTTGGCGGTGACTGTTGCGTTAAGTAA-3' (R449A, D453A)

5'-GGGCCCGCCGCTCGCAGTGGCTCCAGCACA-3' (D460A, R462A)

5'-GCCCTGGCAGGCGGCGGCCAAGTTTGC-3' (H471A, D472A)

5'-GGGGCCTCCCGAAGCGCCCTGGCA-3' (D477A)

5'-CACCAAAGTCATGGCGCCAGCGTTCAGACA-3' D487A, R489A)

5'-CACACCCGGGACAGCCGCCTGTCCACA-3' (K505A, D506A)

5'-GTAGTTGGTAACGGCTGTGTACAC-3' (K513A)

5'-CGGTCGCATGTTGGCAGCAATCCAGGCTAGGTAGTT-3' (D519A, R522A, D523A)

5'-TCCTGGTCACGGTGCCATGTTGGCACGAATCCA-3' (D523A, R526A)

3. Bacterial Transformation and DNA Preparation

The mutant t-PA constructs generated using the protocol above weretransformed into E. coli host strain MM294tonA using the standard CaCl₁procedure (Maniatis et al., supra) for preparation and transformation ofcompetent cells. MM294tonA (which is resistant to T1 phage) was preparedby the insertion and subsequent imprecise excision of a Tn10 transposoninto the tonA gene. This gene was then inserted, using transposoninsertion mutagenesis (Kleckner et al., J. Mol. Biol., 116: 125-159(1977)), into E. coli host MM294 (ATCC 31,446).

DNA was extracted from individual colonies of bacterial transformantsusing the standard miniprep procedure of Maniatis et al., supra. Theplasmids were further purified by passage through a Sepharose CL6B spincolumn, and then analyzed by sequencing and by restriction endonucleasedigestion and agarose gel electrophoresis.

One of these transformants containing the plasmid encoding the K296A,H297A, R298A, R299A mutant, and designated pTPA33-2, was deposited withthe American Type Culture Collection on Jul. 18, 1989 as ATCC No.68,059.

4. Transfection of Human Embryonic Kidney 293 Cells (Small-Scale)

293 cells were grown to 70% confluence in 6-well plates. 2.5 μg of t-PAplasmid DNA mutant was dissolved in 150 μl of 1 mM Tris-HCl, 0.1 mMEDTA, 0.227M CaCl₂. Added to this (dropwise while vortexing) was 150 μlof 50 mM HEPES buffer (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and theprecipitate was allowed to form for ten min. at 25° C. The suspendedprecipitate was then added to the cells in the individual wells in a6-well plate and allowed to settle for four hours in the incubator. Themedium was then aspirated off and 1 ml of 20% glycerol in PBS was addedfor 30 sec. The cells were washed twice, first with 3 ml, then with 1ml, of serum-free medium. Then 3 ml of fresh medium was added and thecells were incubated for five days. The medium was then collected andassayed.

When single-chain t-PA was required, the procedure was as describedabove except that plasminogen-depleted serum was used during the growthphase of the cells.

5. Transfection of Human Embryonic Kidney 293 Cells (Large Scale)

For large-scale purification of the K296A, H297A, R298A, R299A variant,useful for production in significant quantities, the transfectionprocedure used was obtained from Current Protocols in Molecular Biology,Ausubel et al., eds. (Wiley Interscience, 1988) and modified slightly asfollows: A suspension of human embryonic kidney 293 cells was grown in acell culture medium and concentrated by pelleting. The pellet wasresuspended to a concentration of about 10⁸ cells per milliliter and thecells were washed as necessary in serum-free media. The DNA-dextransolution was added at a concentration of about 250 μg of DNA per 500 mlof cells, and this mixture was incubated with mild agitation at 37° C.for up to 90 minutes. DMSO was added to a final concentration of tenpercent and, after about two minutes, fresh medium was added to dilutethe cells to about 10⁶ per milliliter. Cells were then incubated for upto seven days, after which time the supernatant was collected.

Purification of this mutant was accomplished by passage of thesupernatant over a column of glass beads coupled to anti-t-PA goatpolyclonal A6 antibody. The column had been preconditioned with PBS.After the supernatant was loaded, the column was equilibrated with aTris-saline buffer [0.1M Tris.HCl (pH 7.5) and 1M NaCl]. The t-PAvariant was then eluted with 0.1M acetic acid, 0.15M NaCl, 0.02Marginine, and 0.01% Tween 80. Fractions were immediately neutralizedwith Tris base and adjusted to 0.01% Tween 80.

6. Biological Assays

A. t-PA Quantitation

The amount of t-PA present in the cell culture supernatants wasdetermined by the ELISA procedure using polyclonal antibodies preparedagainst wild-type t-PA.

B. S-2288 Assay

The S-2288 assay was used to measure the proteolytic activity of themutants in both the one- and two-chain forms. This assay is a directassay for t-PA proteolytic activity; t-PA cleaves the bond between thesmall peptide and the paranitroanilide chromophore.

Standard curve samples were prepared by diluting wild-type rt-PA withcell culture media. The standard curve samples and rt-PA mutant sampleswere added to the wells of a microtiter plate. If the assay was used tomeasure the activity of two-chain rt-PA, an incubation step with humanplasmin was included in the procedure. Human plasmin (KabiVitrum) wasadded to a final concentration of 0.13 CU (casein units)/ml. The sampleswere incubated for 90 minutes at room temperature. For assaying thesamples in the single-chain form, the plasmin solution was replaced byPBS and the 90-minute incubation was omitted.

Aprotinin [Sigma, approximately 14 TIU (trypsin inhibitor unit)/mg] wasadded to a final concentration of 72 μg/ml to inhibit the plasminactivity, and the samples were incubated at room temperature for 15minutes. A 2.16 mM solution of S-2288 was diluted to 1.45 mM with 0.1MTris, 0.106 mM NaCl, 0.02% sodium azide, pH 8.4, and 100 μl of thissolution was added to each well of the microtiter plate (final volume ineach well was 200 μl). Color development was monitored at 405 nm. Theslope of the absorbance vs. time curve for each standard and sample wasdetermined. A standard curve was prepared by plotting the slope of theabsorbance vs. time curve as a function of rt-PA concentration for thert-PA standards. The relative activity concentration of the mutants wasthen determined from the standard curve. The activity concentration ofeach mutant was divided by the concentration for the mutant obtained inthe rt-PA ELISA, and the resulting specific activities were expressedrelative to wild-type t-PA, which was assigned a value of 1.0.

The data are averages of two assays and are presented as activityrelative to wild-type rt-PA in Table I. The results show that for allmutants presented, the two-chain form is more active (at least 1.5-fold,up to nearly 60-fold) than the one-chain form, relative to wild-typert-PA, indicating that each of these mutants may be considered zymogenicin this assay.

                  TABLE I                                                         ______________________________________                                        Zymogens in the S-2288 Assay                                                              Activity Relative to wt rt-PA                                                 (where wt is 1.0; () indicates                                                experimental error)                                                             one-      two-       fold                                       Mutation      chain     chain      difference                                 ______________________________________                                        R267A         0.33 (0.03)                                                                             0.85 (0.13)                                                                              2.6                                        D283A, H287A  0.08 (0.11)                                                                             0.60 (0.23)                                                                              7.5                                        R339A, R342A  0.01 (0.01)                                                                             0.52 (0.01)                                                                              52                                         E347A, E348A, E349A,                                                                        0.01 (0.01)                                                                             0.49 (0.18)                                                                              49                                         K351A                                                                         K416A, H417A, E418A                                                                         0.01 (0.01)                                                                             0.59 (0.01)                                                                              59                                         E426A, R427A, K429A,                                                                        0.01 (0.01)                                                                             0.33 (0.04)                                                                              33                                         E430A                                                                         H432A, R434A  0.08 (0.01)                                                                             0.71 (0.08)                                                                              8.9                                        R440A         0.53 (0.03)                                                                             0.78 (0.04)                                                                              1.5                                        ______________________________________                                    

C. S-2251 Assay

This assay is an indirect assay for t-PA activity. In this assay,plasminogen is converted to plasmin by the action of t-PA, and plasmincleaves the S-2251 substrate to release the paranitroanilidechromophore. Production of this chromophore is then measured over time.

1. Fibrin-Stimulated S-2251 Assay

Standard curve samples were prepared as described for the S-2288 assay.Samples assayed in the two-chain form were incubated withplasmin-Sepharose. Plasmin-Sepharose was prepared by couplingapproximately 20.8 CU of human plasmin (KabiVitrum) to 1 ml of cyanogenbromide activated Sepharose (Pharmacia). The plasmin-Sepharose (50 μl ofa 5% slurry) was incubated with shaking for 90 min. at room temperaturewith 150 μl of sample. Following the incubation, the resin was removedby centrifugation, and 10 μl of sample were added to the wells of amicrotiter plate.

For samples assayed in the one-chain form, 50 μl of cell culture mediawere added in place of resin, and the incubation step was omitted. Humanthrombin (10 μl of a 42 unit/ml solution) was added to each well. Thereaction in each well was started by the addition of a cocktail (130 μl)composed of 28 μl of human Glu-plasminogen (5.3 μM); 10 μl ofplasminogen-free human fibrinogen (10 μM); 30 μl of 3mM S-2251(KabiVitrum); and 62 μl of PBS. Color development was monitored at 405nm, and the absorbance at the reference wavelength of 492 nm wassubtracted from each time point. The slope of the absorbance vs. timesquared curve was determined for each standard and mutant sample. Astandard curve was prepared by plotting the slope of the absorbance vs.time squared curve as a function of rt-PA concentration for the rt-PAstandards. The determination of the relative specific activity for themutants was as described for the S-2288 assay.

2. Fibrinogen-Stimulated S-2251 Assay

This assay was performed as described for the fibrin-stimulated S-2251assay except that PBS was substituted for the thrombin.

3. Plasma Clot S-2251 Assay

The standard curve sample preparation and the conversion of one-chainrt-PA to two-chain rt-PA using plasmin-Sepharose were as described forthe fibrin-stimulated S-2251 assay. Human thrombin (10 μl of a 31 μg/mlsolution) was added to each well of the microtiter plate. The standardand mutant samples (40 μl) were added to the plate and the reaction wasstarted by adding 100 μl of a mixture of 90 μl of acid citrate dextrosehuman plasma and 10 μl of 9.1 mM S-2251 (KabiVitrum). Color developmentwas monitored at 405 run and the absorbance at the reference wavelengthof 492 nm was subtracted from each time point. The analysis of the datawas as described for the fibrin-stimulated S-2251 assay.

4. Plasma S-2251 Assay

This assay was performed as described for the plasma clot S-2251 assayexcept that PBS was substituted for the thrombin.

Mutants were assayed for zymogenic qualities using the fibrin-dependentand plasma clot-dependent assays, and the results, relative towild-type, are shown in Tables II and III, respectively. Values formutants in the single-chain form are averages of two determinations.Values for mutants in the two-chain form are averages of fourdeterminations.

                  TABLE II                                                        ______________________________________                                        Zymogens in the Fibrin-Dependent S-2251 Assay                                             Activity Relative to wt rt-PA                                                 (where wt is 1.0; () indicates                                                experimental error)                                                             one-      two-       fold                                       Mutation      chain     chain      difference                                 ______________________________________                                        K296A, H297A, R298A,                                                                        1.26 (0.02)                                                                             2.82 (0.32)                                                                              2.2                                        R299A                                                                         E303A, R304A  1.38 (0.04)                                                                             2.13 (0.29)                                                                              1.5                                        H331A, H332A  1.29 (0.04)                                                                             2.19 (0.68)                                                                              1.7                                        R339A, R342A  0.38 (0.07)                                                                             1.04 (0.11)                                                                              2.7                                        K416A, H417A, E418A                                                                         0.21 (0.04)                                                                             0.96 (0.09)                                                                              4.6                                        E426A, R427A, K429A,                                                                        0.14 (0)  0.76 (0.07)                                                                              5.4                                        E430A                                                                         H432A, R434A  0.16 (0.08)                                                                             0.99 (0.13)                                                                              6.2                                        D460A, R462A  0.68 (0.04)                                                                             1.04 (0.08)                                                                              1.5                                        ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        Zymogens in the Plasma Clot-Dependent S-2251 Assay                                        Activity Relative to wt rt-PA                                                 (where wt is 1.0; () indicates                                                experimental error)                                                             one-      two-       fold                                       Mutation      chain     chain      difference                                 ______________________________________                                        D283A, H287A  0.53 (0.01)                                                                             0.82 (0.04)                                                                              1.6                                        K296A, H297A, R298A,                                                                        0.42 (0)  1.29 (0.20)                                                                              3.1                                        R299A                                                                         E303A, R304A  0.90 (0.03)                                                                             1.34 (0.10)                                                                              1.5                                        H331A, H332A  1.09 (0.09)                                                                             1.61 (0.68)                                                                              1.5                                        R339A, R342A  0.19 (0.03)                                                                             0.79 (0.10)                                                                              4.2                                        E347A, E348A, E349A,                                                                        0.38 (0.05)                                                                             0.97 (0.10)                                                                              2.6                                        K351A                                                                         D364A, D365A, D366A                                                                         0.88 (0.03)                                                                             1.46 (0.15)                                                                              1.7                                        K416A, H417A, E418A                                                                         0.19 (0.05)                                                                             0.77 (0.05)                                                                              4.1                                        E426A, R427A, K429A,                                                                        0.11 (0.01)                                                                             0.66 (0.12)                                                                              6                                          E430A                                                                         H432A, R434A  0.05 (0.01)                                                                             0.38 (0.05)                                                                              7.6                                        H445A, R449A   0.80 (ND)*                                                                             1.19 (0.11)                                                                              1.5                                        R449A, D453A  0.79 (0.01)                                                                             1.22 (0.13)                                                                              1.5                                        D460A, R462A  0.21 (0.01)                                                                             0.32 (0.02)                                                                              1.5                                        D477A         0.11 (0.02)                                                                             0.25 (0.08)                                                                              2.3                                        ______________________________________                                         *ND = Not Determined                                                     

A summary of the data in Tables I-III, indicating for which assay eachmutant displays zymogenicity, is shown below, in Table IV:

                  TABLE IV                                                        ______________________________________                                                        Zymogen in                                                                              Fn*     Plasma Clot                                 Mutation          S-2288  2251    2251                                        ______________________________________                                        R267A             X                                                           D283A, H287A      X               X                                           K296A, H297A, R298A, R299A                                                                              X       X                                           E303A, R304A              X       X                                           H331A, H332A              X       X                                           R339A, R342A      X       X       X                                           E347A, E348A, E349A, K351A                                                                      X               X                                           D364A, D365A, D366A               X                                           K416A, H417A, E418A                                                                             X       X       X                                           E426A, R427A, K429A, E430A                                                                      X       X       X                                           H432A, R434A      X       X       X                                           R440A                             X                                           H445A, R449A                      X                                           R449A, D453A                      X                                           D460A, R462A              X       X                                           D477A                             X                                           ______________________________________                                         *Fn = fibrin.                                                            

The zymogenic t-PA variants listed in Table IV were analyzed in theS-2251 fibrin specificity assay and/or S-2251 plasma clot specificityassay in both the one-chain and two-chain forms. The results forone-chain and two-chain t-PA variants are shown in Tables V and VI,respectively.

A summary of the data in Tables V and VI is shown below in Table VII. Itcan be seen that each of the zymogenic rt-PA variants from Table IV isalso fibrin- and/or plasma clot-specific relative to wild-type t-PA. AnX indicates a ratio of fibrin to fibrinogen or plasma clot to plasmaof >1.5 as measured in the S-2251 assay and reported in Tables V and VI.

                                      TABLE V                                     __________________________________________________________________________    The activities are relative to wt rt-Pa (where wt is 1.0).                    FIBRIN- AND PLASMA CLOT-SPECIFICITY OF rt-Pa VARIANTS                         (ONE-CHAIN)                                                                   Mutant    Fg   Fb   Fb/Fg                                                                              Pl   PC   PC/Pl                                      __________________________________________________________________________    AVE  R267A                                                                              0.53 0.92 1.74 0.24 0.71 3.00                                       SD        (0.00)                                                                             (0.01)    (0.01)                                                                             (0.09)                                          AVE  D283A,                                                                             0.59 0.78 1.32 0.29 0.53 2.76                                       SD   E287A                                                                              (0.02)                                                                             (0.05)    (0.07)                                                                             (0.01)                                          AVE  K296A,                                                                             0.29 1.26 4.40 0.14 0.42 3.00                                       SD   B297A,                                                                             (0.05)                                                                             (0.02)    (0.03)                                                                             (0.00)                                               R296A,                                                                        R299A                                                                    AVE  E303A,                                                                             0.68 1.38 2.04 0.35 0.90 2.61                                       SD   R304A,                                                                             (0.09)                                                                             (0.04)    (0.01)                                                                             (0.03)                                          AVE  H331A,                                                                             1.19 2.29 1.08 1.12 1.09 0.97                                       SD   H332A                                                                              (0.06)                                                                             (0.04)    (0.13)                                                                             (0.09)                                          AVE  R339A,                                                                             0.34 0.38 1.12 0.02 0.19 9.50                                       SD   R342A                                                                              (0.00)                                                                             (0.07)    (0.03)                                                                             (0.03)                                          AVE  E347A,                                                                             0.50 0.87 1.75 0.13 0.38 2.88                                       SD   E348A,                                                                             (0.09)                                                                             (0.15)    (0.04)                                                                             (0.05)                                               E349A,                                                                        K351A                                                                    AVE  D364A,                                                                             0.66 1.50 2.27 0.28 0.88 3.14                                       SD   D365A,                                                                             (0.03)                                                                             (0.04)    (0.04)                                                                             (0.03)                                               D366A                                                                    AVE  K416A,                                                                             0.34 0.21 0.60 0.10 0.19 1.95                                       SD   H417A,                                                                             (0.01)                                                                             (0.04)    (0.04)                                                                             (0.05)                                               E418A                                                                    AVE  E426A,                                                                             0.21 0.14 0.68 0.07 0.11 1.50                                       SD   R427A,                                                                             (0.01)                                                                             (0.00)    (0.01)                                                                             (0.01)                                               K429A,                                                                        E430A                                                                    AVE  H432A,                                                                             0.16 0.16 1.00 0.08 0.05 0.60                                       SD   R434A                                                                              (0.01)                                                                             (0.08)    (0.01)                                                                             (0.01)                                          AVE  R440A                                                                              0.62 1.02 1.64 0.48 0.86 1.81                                       SD        (0.08)                                                                             (0.18)    (0.04)                                                                             (0.10)                                          AVE  H445A,                                                                             0.52 1.12 2.15 0.24 0.80 3.33                                       SD   R449A                                                                    AVE  R449A,                                                                             0.58 1.16 2.00 0.28 0.79 2.87                                       SD   D453A                                                                              (0.01)                                                                             (0.11)    (0.01)                                                                             (0.01)                                          AVE  D460A,                                                                             0.13 0.68 5.19 0.10 0.21 2.16                                       SD   R462A                                                                              (0.03)                                                                             (0.04)    (0.03)                                                                             (0.01)                                          AVE  D477A                                                                              0.08 0.09 1.06 0.03 0.11 4.20                                       SD        (0.00)                                                                             (0.01)    (0.04)                                                                             (0.02)                                          __________________________________________________________________________     Fg = Fibrinogen                                                               Fb = Fibrin                                                                   Pl = Plasma                                                                   PC = Plasma clot                                                              AVE = Average                                                                 SD = Standard deviation                                                  

                                      TABLE VI                                    __________________________________________________________________________    The activities are relative to wt rt-Pa (where wt is 1.0).                    FIBRIN AND PLASMA CLOT SPECIFICITY OF rt-PA VARIANTS                          (TWO-CHAIN)                                                                   Mutant    Fg   Fb   Fb/Fg                                                                              Pl   PC   PC/Pl                                      __________________________________________________________________________    AVE  R267A                                                                              0.85 0.97 1.14 0.70 0.86 1.23                                       SD        (0.21)                                                                             (0.15)    (0.08)                                                                             (0.11)                                          AVE  D283A,                                                                             0.77 1.00 1.31 0.69 0.82 1.19                                       SD   H287A                                                                              (0.12)                                                                             (0.08)    (0.14)                                                                             (0.04)                                          AVE  K296A,                                                                             0.31 2.92 9.24 0.24 1.29 5.50                                       SD   H297A,                                                                             (0.14)                                                                             (0.32)    (0.15)                                                                             (0.20)                                               R298A,                                                                        R299A                                                                    AVE  E303A,                                                                             0.56 2.13 3.79 0.26 1.34 5.10                                       SD   R304A                                                                              (0.23)                                                                             (0.29)    (0.08)                                                                             (0.10)                                          AVE  H331A,                                                                             1.03 2.19 2.12 1.03 1.61 1.56                                       SD   H332A                                                                              (0.86)                                                                             (0.68)    (0.67)                                                                             (0.68)                                          AVE  R339A,                                                                             0.55 1.04 1.89 0.23 0.79 3.38                                       SD   R342A                                                                              (0.18)                                                                             (0.11)    (0.05)                                                                             (0.10)                                          AVE  E347A,                                                                             0.76 1.33 1.76 0.44 0.97 2.20                                       SD   E348A,                                                                             (0.24)                                                                             (0.12)    (0.11)                                                                             (0.10)                                               E349A,                                                                        K351A                                                                    AVE  D364A,                                                                             0.73 1.77 2.44 0.26 1.46 5.68                                       SD   D365A,                                                                             (0.17)                                                                             (0.23)    (0.12)                                                                             (0.15)                                               D366A                                                                    AVE  K416A,                                                                             0.77 0.96 1.24 0.42 0.77 1.82                                       SD   H417A,                                                                             (0.13)                                                                             (0.09)    (0.09)                                                                             (0.05)                                               E418A                                                                    AVE  E426A,                                                                             0.38 0.75 2.01 0.24 0.66 2.80                                       SD   R427A,                                                                             (0.27)                                                                             (0.07)    (0.07)                                                                             (0.12)                                               K429A,                                                                        E430A                                                                    AVE  H432A,                                                                             0.16 0.99 6.40 0.16 0.38 2.31                                       SD   R434A                                                                              (0.05)                                                                             (0.13)    (0.05)                                                                             (0.05)                                          AVE  R440A                                                                              0.51 0.92 1.81 0.51 0.90 1.78                                       SD        (0.11)                                                                             (0.10)    (0.08)                                                                             (0.09)                                          AVE  H445A,                                                                             0.62 1.46 2.36 0.29 1.19 4.06                                       SD   R449A                                                                              (0.16)                                                                             (0.10)    (0.09)                                                                             (0.11)                                          AVE  R449A,                                                                             0.74 2.50 2.04 0.36 1.22 3.36                                       SD   D453A                                                                              (0.14)                                                                             (0.23)    (0.12)                                                                             (0.13)                                          AVE  D460A,                                                                             0.18 1.04 5.91 0.12 0.32 2.59                                       SD   R462A                                                                              (0.12)                                                                             (0.08)    (0.02)                                                                             (0.02)                                          AVE  D477A                                                                              0.12 0.10 0.89 0.11 0.25 2.24                                       SD        (0.07)                                                                             (0.02)    (0.03)                                                                             (0.08)                                          __________________________________________________________________________     Fg = Fibrinogen                                                               Fb = Fibrin                                                                   Pl = Plasma                                                                   PC = Plasma clot                                                              AVE = Average                                                                 SD = Standard deviation                                                  

                  TABLE VII                                                       ______________________________________                                                       Fibrin    Plasma Clot                                                         Specificity                                                                             Specificity                                          Mutant           1-chain 2-chain 1-chain                                                                             2-chain                                ______________________________________                                        R267A            X               X                                            D283A, H287A                     X                                            K296A, H297A, R298A, R299A                                                                     X       X       X     X                                      E303A, R304A     X       X       X     X                                      H331A, H332A             X             X                                      R339A, R342A             X       X     X                                      E347A, E348A, E349A, K351A                                                                     X       X       X     X                                      D364A, D365A, D366A                                                                            X       X       X     X                                      K416A, H417A, E418A              X     X                                      E426A, R427A, K429A, E430A                                                                             X       X     X                                      H432A, R434A             X             X                                      R440A            X       X       X     X                                      H445A, R449A     X       X       X     X                                      R449A, D453A     X       X       X     X                                      D460A, R462A     X       X       X     X                                      D477A                            X     X                                      ______________________________________                                    

There was a striking correlation between those variants of rt-PAexhibiting fibrin and/or plasma clot specificity and those that meet thecriteria of zymogenicity specified above. In addition, two variants ofrt-PA were found to exhibit fibrin specificity but were not zymogenicrelative to wild-type rt-PA. Table VIII shows the S-2251 assay data forthe fibrin-specific and plasma clot-specific variants.

                                      TABLE VIII                                  __________________________________________________________________________    FIBRIN- AND PLASMA CLOT-SPECIFIC, NON-ZYMOGENIC                               VARIANTS OF rt-PA                                                                 Mutant                                                                             Fg/1ch                                                                            Fb/1ch                                                                             Fb/Fg1ch                                                                            Pl/1ch                                                                            PC/1ch                                                                             PC/P11ch                                     __________________________________________________________________________    AVE E408A                                                                              0.35                                                                              0.79 2.28  0.19                                                                              0.47 2.51                                         SD       (0.01)                                                                            (0.06)     (0.04)                                                                            (0.01)                                            AVE E410A                                                                              0.61                                                                              0.90 1.47  0.51                                                                              0.74 1.44                                         SD       (0.08)                                                                            (0.04)     (0.11)                                                                            (0.05)                                            __________________________________________________________________________        Mutant                                                                             Fg/2ch                                                                            Fb/2ch                                                                             Fb/Fg2ch                                                                            Pl/2ch                                                                            PC/2ch                                                                             PC/P12ch                                     __________________________________________________________________________    AVE E408A                                                                              0.34                                                                              0.89 2.61  0.24                                                                              0.64 2.72                                         SD       (0.18)                                                                            (0.11)     (0.08)                                                                            (0.04)                                            AVE E410A                                                                              0.55                                                                              0.92 1.67  0.48                                                                              0.88 1.84                                         SD       (0.13)                                                                            (0.09)     (0.10)                                                                            (0.08)                                            __________________________________________________________________________     Fg = Fibrinogen                                                               Fb = Fibrin                                                                   Pl = Plasma                                                                   PC = Plasma clot                                                              1ch = 1 chain                                                                 2ch = 2 chain                                                                 AVE = Average                                                                 SD = Standard deviation                                                  

Deposit of Materials

The following culture has been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

    ______________________________________                                        Strain         ATCC Dep. No.                                                                              Deposit Date                                      ______________________________________                                        pTPA33-2 in    68,059       July 18, 1989                                     E. coli MM294tonA                                                             ______________________________________                                    

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromthe date of deposit. The organism will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweenGenentech, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the culture to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC§122 and the Commissioner's rules pursuant thereto (including 37 CFR§1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureon deposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofone aspect of the invention and any constructs that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustration that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art-from the foregoing description andfall within the scope of the appended claims.

What is claimed is:
 1. A method for identifying tissue plasminogenactivator (t-PA) variants having one or more of the followingproperties: zymogenic character, fibrin specificity, or plasma clotspecificity as compared to the corresponding wild-type t-PA,comprising(a) substituting at least one amino acid within the range ofamino acids 303-304 of the amino acid sequence of the correspondingwild-type human t-PA, and (b) screening the resultant t-PA variant forone or more of the following biological properties: zymogenic character,fibrin specificity, and plasma clot specificity.
 2. The method of claim1 wherein an amino acid is substituted at amino acid position
 303. 3.The method of claim 1 wherein an amino acid is substituted at amino acidposition
 304. 4. The method of claim 1 wherein an amino acid issubstituted at each of amino acid positions 303 and
 304. 5. The methodof claim 4 wherein each of amino acid positions 303 and 304 issubstituted by alanine.
 6. The method of claim 1 wherein the amino acidused for substitution is selected from the group consisting of alanine,serine, threonine, asparagine, glutamine, phenylalanine, and tyrosine.7. The method of claim 6 wherein the amino acid used for substitution isselected from the group consisting of alanine, serine and threonine. 8.The method of claim 1 wherein the resultant t-PA variant is screened forzymogenic character.
 9. The method of claim 1 wherein the resultant t-PAvariant is screened for fibrin specificity.
 10. The method of claim 1wherein the resultant t-PA variant is screened for plasma clotspecificity.